Synthesis of Compound Giant Unilamellar Vesicles: A Biomimetic Model of Nucleate Cells

Summary

This article presents a method for preparing compound Giant Unilamellar Vesicles (cGUVs) with a vesicle-in-vesicle structure, with customized electrical conductivity in inner, annular, and outer regions. Electroformation synthesizes simple GUVs, which are transformed into stomatocytes and cGUVs via osmotic shock, providing a valuable model for studying the biophysics of nucleated cells.

Abstract

A simple Giant unilamellar vesicle (sGUV) is a micron-sized spherical vesicle, which is composed of a lipid bilayer that encloses an aqueous solution inside and is suspended in another aqueous medium. As biomimetic models of biological cells, sGUVs are well-established systems for biophysical studies. However, sGUVs primarily mimic anucleate cells, limiting their application as models for non-nucleate cells. We propose compound Giant Unilamellar vesicles (cGUVs) as a more appropriate biomimetic model for nucleated cells. cGUVs are vesicle-in-vesicle structures, where the outer and inner vesicle bilayers can be assumed to represent the cell and the nuclear membrane of a cell, respectively.

In this study, we describe a simple method for the synthesis of cGUVs. Briefly, sGUVs were generated using the lipid composition of DMPC and cholesterol within the range of 37-43 mol% in a sucrose medium via the electroformation method. These sGUVs were then subjected to osmotic shock by introducing a hypertonic glucose solution, triggering an immediate transition to a somatocytic shape. This process led to the formation of an intermediate state where the outer and inner vesicles remained connected through a neck, ultimately resulting in the development of vesicle-in-vesicle structure, referred to as cGUVs.

Furthermore, it is known that GUVs prepared with higher cholesterol levels in the bilayer exhibit reduced ion permeability. This characteristic allows tuning of conductivity in the annular solution of a cGUV by adjusting the conductivities of the hydrating medium, the inner solution through hypertonic glucose, and the outer solution by adjusting of the dilution or the addition of conductive media, making the resultant cGUVs with controlled conductivities in the outer, annular, and inner regions, relevant for electric field studies. We propose this efficient and straightforward approach for synthesizing compound vesicles that can be used as a biomimetic model for eukaryotic, nucleated cells, advancing their application in biophysical research.

Introduction

Biomimetic models have been extensively employed to study the complex biophysical phenomena of cell membranes. These simplest biomimetic models provide valuable insights by minimizing interference from the myriad variables present in biological cells. Giant Unilamellar Vesicles (GUVs) are a foundational biomimetic system used to study membrane biophysics^1,2. GUVs are well established as a representative model of the cell membrane. They comprise a single spherical lipid bilayer of size 10-30 µm in diameter and are encapsulated and suspended in an aqueous solution. In this article, we term these sGUVs. These systems are constructed using a bottom-up approach, where essential cellular components -- lipids, proteins, and catalysts -- are assembled to mimic the structure and function of biological membranes^3,4.

Although simple sGUVs, with only the aqueous core inside, are commonly used, more advanced models have been developed to replicate specific features of biological cells. For example, GUVs with actin-supported structures emulate the cytoskeleton^5,6,7, while high-salt environments are used to imitate physiological conditions. The higher salt environments mimic physiological conditions but it is difficult to obtain them in GUVs since traditional methods, such as electroformation^8,9, are often unsuitable for synthesizing micron-sized GUVs under such conditions. Newer methods, such as the gel-assisted method, which uses polymeric or starch gels to aid bilayer swelling during hydration, address these limitations^10,11. Similarly, morphological changes of the sGUVs have been studied¹². The addition of closed and open filaments, such as encapsulated actin, microtubules, and other cytoskeletal components, of varying rigidity, and the osmotic stress condition inside them influences the morphological transition of these osmotically deflated but initially quasi-spherical vesicles. Theoretical analysis reveals diverse morphological transformations, including tubular, spherical, racket-like, cherry-like, and droplet structures, aligning well with experimental observations¹². These findings have significant implications for understanding the organization of filaments, microtubules, and nanorings within cells and vesicles. Other studies review methods for protein encapsulation and explore how encapsulated actin, microtubules, and cytoskeletal components regulate the shape of the osmotic deflated vesicles^12,13.

Further, inspired by eukaryotic cell architecture, complex GUV-based models are developed towards the development of artificial models resembling fission and cell division, reproduction and multicompartment features of eukaryotic cells, self-reproducing cells^14,15,16,17, and multicompartment cells. Beyond structural application, GUVs are also the chosen models for understanding cell electroporation -- a process where an applied electric field induces pore formation in the lipid membrane due to Maxwell stress. GUVs, with dimensions comparable to biological cells, exhibit electrodeformation and electroporation under electric fields. Such studies on GUVs provide insights into electromechanical properties such as membrane capacitance and bending rigidity¹⁸ and into electroporation mechanisms^2,19,20. However, practically, these studies are best representative of anucleate cells, such as red blood cells (RBCs).

To bridge this gap, we propose compound Giant Unilamellar Vesicles (cGUVs) as a novel biomimetic model for nucleate eukaryotic cells^21,22. These vesicles possess a vesicle-in-vesicle structure, where the inner bilayer mimics the nuclear membrane, and the outer bilayer represents the plasma membrane. Furthermore, we demonstrate how the electrical conductivity of the inner, annular, and outer regions can be modulated without requiring advanced instrumentation, such as micromanipulators or microsyringes. This simplified approach to synthesizing cGUVs offers a promising platform for advancing biophysical research on nucleated cells.

Protocol

1. Preparation of lipid mixture solutions

1. Prepare 1 mL solution of DMPC and cholesterol in a 63:37 molar ratio at a final concentration of 5 mg/mL. For this, add 149 µL of DMPC (25 mg/mL stock as received dissolved in chloroform), 252 µL of cholesterol (5 mg/mL cholesterol dissolved in chloroform), and 599 µL of chloroform.
2. Prepare another 1 mL solution of DMPC: Cholesterol in a 57:43 molar ratio at 5 mg/mL, mix 139.8 µL of DMPC (25 mg/mL stock as received dissolved in chloroform), 301 µL of cholesterol (5 mg/mL, prepared from powder dissolved in chloroform), and 559.2 µL of chloroform.
3. To stain the lipid bilayer, add 100 µL of Nile Red (from a 5 mM stock solution) to 1 mL of the lipid mixture containing DMPC and cholesterol in a 63:37 molar ratio (described in steps 1.1 and 1.2). Alternatively, add 30 µL of Liss Rhod-PE (1mg/mL stock of Liss Rhod-PE) to the 1 mL of prepared lipid mixture (described in steps 1.1 and 1.2).
   NOTE: As the solvent (chloroform) is highly volatile, after repeated opening of the vial for use of the stock solution (25 mg/mL of DMPC), it may get concentrated, which may change the composition.

2. Electroformation protocol for synthesizing simple Giant Unilamellar Vesicles (sGUVs)

1. Thoroughly clean Indium Tin Oxide (ITO) coated slides with a dish soap solution and rinse with deionized water (0.055 µS/cm). Clean the slides with 100% ethanol solution, rinse with deionized water, and dry in an oven at 85 °C.
2. Identify the conductive side of the ITO-coated slides using a Clampmeter. Secure the cleaned ITO-coated slide on the spin coater stage using a vacuum with the conductive side facing up.
3. Apply 25 µL of the lipid solutions (prepared in step 1.1) by casting four droplets of the solution onto the conductive side of the ITO slide. Then, take another ITO slide and apply 25 µL of the lipid solutions (prepared in step 1.2) by casting four droplets of the solution onto the conductive side of the ITO slide.
4. Vacuum-dry both lipid-coated slides in a desiccator that is kept in the dark for at least 2 h.
   NOTE: Protection from light protects the lipids from possible chemical changes.
5. Arrange a lipid-coated ITO slide (coated with lipid prepared in step 1.1) in parallel with other clean ITO slides, conductive side facing each other, with a 3 mm-thick silicone rubber spacer between them. Seal the assembly with a clamp to create a secure electroformation chamber.
6. Follow the same procedure described in step 2.5 for the ITO-coated slide with lipid solution prepared in step 1.2 to form an electroformation chamber.
7. Place both chambers in an incubator maintained at 38-40 °C and fill the chamber with 100 mM of sucrose solution with the help of a 2 mL syringe. Apply an AC electric field of 5 Vpp at 10 Hz frequency using a dual-channel function generator for 4 h.
   NOTE: Ensure the ITO slide's conductive side is correctly clamped to the crocodile clips that connect with the function generator.
8. Disconnect the electroformation chamber from the function generator. Remove the chamber from the incubator and allow it to cool to room temperature. Harvest the synthesized sGUVs from both chambers using a syringe and transfer them each into a 2 mL microcentrifuge tube and incubate at room temperature (25-27 °C) for 1 h before subjecting it to osmotic shock.

3. Shape transition in osmotic shock (sGUV-to-Stomatocyte-to-cGUV)

1. Transfer 200 µL of sGUVs suspended in hydrating media containing 100 mM sucrose solution to an observation chamber.
2. Introduce 125 µL of glucose solution (300 mM) to this sGUV suspension containing 200 µL of 100 mM sucrose within the observation chamber to induce osmotic shock that initiates the shape transition of the sGUVs.
   NOTE: Follow the same procedure (given in step 3) for sGUV synthesis from the lipid composition given in steps 1.1 and 1.2 for osmotic shock-driven shape transition.
3. Allow the osmotic shock-induced sGUVs to rest for 1 h in the observation chamber placed on the microscopy stage.
4. To conduct and observe the shape transition of sGUVS, place the observation chamber (electrofusion chamber, a Petri dish, a concavity slide, or any other chamber that can be used for microscopy observation) on the stage of an inverted microscope and allow the vesicles to settle.
5. Monitor the shape transitions occurring within the chamber. Use Differential Interference Contrast (DIC) and epifluorescence microscopy with Plan fluor, ELWD 20x/0.45, and Plan fluor, ELWD 40x/0.60 objectives lens to observe the shape transitions of the sGUVs.
6. Observe the formation of stomatocytic vesicles during transition. Monitor the chamber over time as larger vesicles settle at the bottom, followed by an increased number of smaller vesicles.

4. Modifying conductivity in the inner, annular, and outer regions of cGUVs

1. Adjust the conductivity of solutions in different regions of cGUVs (inner, annular, and outer) by adding appropriate quantities (µL) of a 7.5 mM stock salt solution before giving an osmotic shock to sGUVs.
   1. For example, to create higher conductivity in the outer and inner regions compared to the annular region, transfer 200 µL of sucrose solution into the electrofusion chamber. Add 20 µL of 7.5 mM salt solution to the chamber. Induce osmotic shock by adding 125 µL of 300 mM glucose solution.
   2. Allow the osmotic-shocked sGUVs to rest for 3-4 h. In this case, cGUVs are obtained with higher conductivity in the outer and inner regions than in the annular region at the end.
2. Perform electrodeformation of cGUVs by applying an AC electric potential of 7.5 Vpp at 100 kHz using a function generator in an electrofusion chamber that contains wire electrodes spaced 500 µm apart.
3. After applying an AC electric field, capture video at 10 frames/s and observe the oblate shape deformation of the outer vesicles and prolate shape deformation of the inner vesicles, confirming higher conductivity in the outer and inner regions than in the annular region in cGUV domains.

5. Microscopy analysis of cGUVs

1. Inverted Microscope Imaging: Perform differential interference contrast (DIC) and epifluorescence microscopy using an inverted microscope and coupled with a monochromatic camera. Use DIC and epifluorescence microscopy with Plan fluor, ELWD 20x/0.45 and Plan fluor, ELWD 40x/0.60 objective lenses to observe the shape transitions of the sGUVs. Use a green filter (EX 510-560, DM 575, BA 590) for the Nile red stain bilayer for the epifluorescence study.
   NOTE: DIC images provide valuable insights into the status of the connected neck, confirming whether it remains intact or undergoes fusion to form cGUVs.
2. Use ImageJ software to insert a scale bar and crop microscopy images. To add a scale bar, go to Analyze | Set Scale to calibrate the image, then go to Analyze | Tools | Scale Bar to insert the scale bar.
3. Confocal Microscopy for morphology: To obtain a clearer understanding of the cGUV morphology, a laser scanning confocal microscope is employed for z-axis scanning with a step size of 1 µm. Imaging with Confocal Microscope: use a laser scanning confocal microscope with a Plan-Apochromat 40x/1.3 oil DIC objective lens to image the cGUVs. Image Nile Red or Rhodamine-PE stained cGUVs using a red single-channel mode, with an excitation wavelength of 561 nm and an emission range of 561-695 nm.
4. To modify and extract the .czi file of the image in the microscope-linked software, insert graphics like a scale bar and enable 2D, 3D view, then go to Processing | Method | Parameters and click Apply to get an image in JPG format.
5. Preparation for Confocal Microscopy: fill an aqueous solution containing cGUVs in a modified custom chamber (bottom glass replaced with Coverslip glass) for confocal imaging. Leave the sample undisturbed for 10-15 min to allow the cGUVs to settle for confocal imaging; load the cGUVs mixture in the cavity slide and seal with a coverslip to arrest the movements of cGUVs. Following this, analyze the cGUVs through confocal microscopy and z-scanning with 1 µm intervals.

Results

sGUVs were synthesized using the electroformation method (as shown in Figure 1) and harvested from the chamber using a syringe. The harvested sGUVs were initially visualized under an inverted microscope and subjected to an osmotic shock to induce their transformation into a stomatocyte shape. After further membrane remodeling, this stomatocyte is transformed into a vesicle-in-vesicle structure called a compound Giant Unilamellar Vesicle (cGUV) (Figure 1). The cGUVs were further analyzed using confocal microscopy (Figure 2), where Differential Interference Contrast (DIC) and fluorescence imaging were employed. A 3D reconstruction of the cGUVs was created through z-scanning at 1 µm intervals. The 3D images clearly showed that the inner vesicle was separated entirely from the outer vesicle and positioned at an elevated z-plane. This position of the inner vesicles is attributed to the lower density of the inner vesicular solution (sucrose + glucose) compared to the annular region solution (sucrose).

In the intermediate state, when the sGUV transitions into a stomatocyte shape, the outer solution is engulfed and becomes part of the inner solution within the inner vesicle. However, the inner and outer vesicles remain connected through a narrow neck (Figure 3 and Figure 4). This intermediate stage is visualized under a confocal microscope in a settled cGUV, where the connected neck is observed in the lower z-plane. A 3D image confirms the neck's position during this transitional state (Figure 4). Over approximately 4-6 h, the narrow neck connecting the outer and inner vesicles fuses, leading to their complete separation. There are cases also where narrow necks get fused instantly. However, the duration required for neck fusion varies in cGUVs. Notably, cGUVs subjected to overnight osmotic shock consistently show a complete fusion of the neck connections, with the inner and outer vesicles fully separated.

The overall population of osmotic shock-induced sGUVs transitioning into cGUVs is presented in Figure 5. A variety of shapes, including star-like structures, multiple inner vesicles, tubules, and distorted spherical forms, were observed in addition to cGUVs. Figure 5 shows a DIC and fluorescent image captured at 20x magnification, 6 h after the osmotic shock was applied to the sGUVs. Figure 6 depicts the cGUVs population formed with a lipid composition of DMPC: Chol (63:37), and shows that a significant population of cGUVs is formed when DMPC: Chol is used in the molar ratio of 63:37. Figure 7 presents electrodeformation of the cGUV under an AC electric field, where Figure 7(ii) shows an oblate and prolate shape deformation for outer and inner vesicles, respectively.

Figure 1: Schematic representation: (i) Electroformation chamber consisting of two parallel ITO-coated slides separated by a Teflon spacer, (ii) Harvested sGUVs suspended in a sucrose-based hydrating medium, the conductivity of the hydrating media remains consistent for the annular solution (iii) Formation of stomatocyte shapes in response to osmotic shock applied to sGUVs, the conductivity of inner solution based on the conductivity of the glucose solution used for osmotic shock, and (iv) Transition of stomatocyte shapes into cGUVs, (v) dilution of cGUV in sucrose+glucose with same osmolarity to lower the outer solution conductivity or addition of salt solution to increase the conductivity of the outer solution. Abbreviations: ITO = Indium Tin Oxide; Su = Sucrose; Glu = Glucose; sGUV =simple Giant Unilamellar Vesicle; cGUV = compound Giant Unilamellar Vesicle; IS = Inner Solution; AS = Annular Solution; OS = Outer Solution. Please click here to view a larger version of this figure.

Figure 2: Confocal microscopy images of a cGUV: (i) Differential interference contrast image of the cGUV in the equatorial plane, showing its maximum size; (ii) corresponding fluorescence image of the cGUV; (iii) three-dimensional reconstruction of the cGUV obtained through z-axis scanning. Scale bars = 20 µm. Abbreviation: cGUV = compound Giant Unilamellar Vesicle. Please click here to view a larger version of this figure.

Figure 3: Intermediate stage preceding cGUV formation. (i) Differential interference contrast image at a lower z-position, highlighting the neck structure; (ii) corresponding fluorescence image; (iii) differential interference contrast image in the equatorial plane; and (iv) fluorescence image at the equatorial plane. Scale bars = 20 µm. Abbreviation: cGUV =compound Giant Unilamellar Vesicle. Please click here to view a larger version of this figure.

Figure 4: 3D reconstructed images of the intermediate stage. (i) and (ii) depict the connected neck position (indicated with arrowhead) between the inner and outer vesicles of the same cGUV, shown at different rotation angles. Abbreviation: cGUV = compound Giant Unilamellar Vesicle. Please click here to view a larger version of this figure.

Figure 5: Population of cGUVs formed after osmotic shock applied to sGUVs: (i) Differential Interference contrast image and (ii) corresponding epifluorescence image. Scale bar = 50 µm. Abbreviations: cGUV =compound Giant Unilamellar Vesicle; sGUV = simple Giant Unilamellar Vesicle; DMPC = 1,2-dimyristoyl-sn-glycero-3-phosphocholine; CHOL =Cholesterol. Please click here to view a larger version of this figure.

Figure 6: A significantly large number of cGUVs syntheses with DMPC/CHOL in a molar ratio of 63:37 after 8 h of osmotic shock. Scale bar = 30 µm. Abbreviations: cGUV = compound Giant Unilamellar Vesicles; DMPC =1,2-dimyristoyl-sn-glycero-3-phosphocholine; CHOL =Cholesterol. Please click here to view a larger version of this figure.

Figure 7: cGUV electrodeformation under an AC electric field: (i) Undeformed cGUV before applying an AC electric field, (ii) Steady state deformation of cGUV under an AC electric field. Notice the oblate shape of the outer and prolate shape of the inner vesicle. Applied electric field parameters (electric field amplitude = 7.5 Vpp and frequency 100 kHz). Scale bar = 30 µm. Abbreviations: cGUV = compound Giant Unilamellar Vesicle; AC = Alternating current. Please click here to view a larger version of this figure.

Discussion

The composition and types of lipids largely determine the mechanical properties of the bilayer constituting a GUV. A bilayer composed of DMPC/Chol exhibits unique shape transitions when subjected to osmotic shock. Under a strong hypertonic osmotic shock, the aqueous fluid inside the sGUV is expelled, leading to the deformation of the GUV bilayer. The observed shape transitions are influenced primarily by the outward flux of water and the bilayer's bending rigidity. These deformations manifest in various forms, including starfish shapes and dumbbells, depending on the strength of the osmotic shock and the lipid composition of the bilayer.

Interestingly, the intrinsic properties of the DMPC-Cholesterol bilayer are favorable to induce a transition of the sGUV into a stomatocyte shape at room temperature. During this transition, the narrowing of the neck region results in vesicle-in-vesicle (VIV) formation. In the final VIV structure, the inner vesicles are completely separated from the outer vesicles. Previous studies have reported that for a bilayer composition of DMPC/Chol in a 70:30 molar ratio, complete separation of inner vesicles occurs within approximately 30 s when vesicles are synthesized in pure water and glucose is used for inducing an osmotic shock¹⁵. In contrast, we synthesized sGUVs with a composition of 67:37 DMPC: Chol molar ratio in a sucrose solution to create a density difference with the addition of glucose in the outer suspension, allowing them to settle at the bottom of the chamber for easier microscopic observation. Additionally, it is observed that the sGUV synthesis with a cholesterol content of lower than 30% molar ratio shows higher permeability and a decrease in the contrast of sucrose and glucose, which makes them difficult to settle at the bottom for microscopy observation. Under these conditions, we observed that the complete separation of the inner vesicle from the outer vesicle typically occurs over approximately 4-5 h. However, depending on the intrinsic membrane properties, it can take less or more time. Furthermore, when the obtained GUVs are left overnight after osmotic shock, nearly all VIV-shaped vesicles exhibit complete separation of the inner vesicle.

It should be mentioned that a significant percentage of vesicles transition into a VIV structure. At the same time, other shapes, such as dumbbells and distorted spheres, are also observed following the osmotic shock. Large sGUVs with a radius greater than 20 µm rarely transition into a stomatocyte shape. Instead, these sGUVs typically remain deformed in deflated non-spherical shapes, or nanotubules engulfed inside the GUV to release bending energy. The process of transformation of sGUV into cGUV proceeds as follows. When a freshly prepared sGUV sample (37 mol % cholesterol) is allowed to rest for an hour and then an osmotic shock is applied to this sGUV solution, an instantaneous transition stomatocytes is observed, and the vesicles showing VIV structure start to settle down at the bottom of the chamber; waiting for 2 h shows a significant population of VIV structure vesicles settled at the bottom of chamber. A mixed population was observed in these VIV transit vesicles. In most vesicles showing VIV structure, the outer and inner vesicles were completely separated, while in a few, the inner and outer vesicles remained attached through a neck.

In some instances, it is possible that freshly prepared GUVs cannot transit to the stomatocytic shape under osmotic shock. These cases are typically observed at 43 mol% cholesterol. In these cases, the sGUV samples were allowed to rest for more than 3-4 h at room temperature, followed by the application of osmotic shock, leading to a stomatocyte shape transition. Notably, the properties of the bilayer appear to undergo change over time, explaining the different rest times required for the two sGUVs of two different DMPC:Chol compositions to form stomatocytes and thereafter, cGUVs. The bilayers with lower cholesterol content (37 mol%) exhibit lower bending rigidity compared to those with higher cholesterol content (43 mol%). Bending rigidity tends to increase with cholesterol concentration, particularly when examined at temperatures above the melting transition temperature of DMPC (T_m = 24 °C)²³. In the case of pure DMPC bilayer, a notable drop in bending rigidity occurs at the transition temperature (T_m), where the bilayer enters the ripple phase. In this phase, lipid molecules adopt a tilted packing configuration, reducing bending rigidity. Upon further increasing the temperature (T_m + 2 K), the bending rigidity rises to a specific value and stabilizes, showing only minor variations with additional temperature increases. This observation suggests that above the transition temperature, in the fluid state, the bending rigidity of the DMPC bilayer remains relatively constant²⁴. A similar trend has been reported for bilayers composed of DMPC and lower cholesterol concentrations (<12.5%), and a tilted lipid packing is reported near the phase transition temperature of DMPC^23,25. The existing literature states that the bending rigidity of bilayers containing more than 30 mol% cholesterol has higher bending rigidity at (T > T_m) but does not compare bending rigidity fluctuations around T_m at such higher cholesterol content²³. Based on our experimental observations, GUVs composed of DMPC with 37-43 mol% cholesterol have suitable bending rigidity to deform GUVs into a stomatocyte shape at room temperature (~25-27 °C) under osmotic stress. GUVs incubated at T > 35 °C show tiny multiple vesicles engulfed inside the GUVs (data not shown).

Based on the DMPC/Chol phase diagram, the sGUV bilayer composition, which contains 37-43 mol% cholesterol, remains in the liquid-ordered (Lo) phase below and above the phase transition temperature T_m^26,27. The presence of this phase is consistent with our observed membrane properties, including reduced membrane permeability and enhanced mechanical stability^28,29. In Figure 7, the oblate and prolate deformation of the cGUV indicates that the outer and inner regions exhibit higher conductivity compared to the annular region. This confirms that the conductivity differences within the cGUV persist over an extended period. For a more detailed investigation of cGUV electrodeformation, refer to our previous work²¹. The low permeability of the membrane assists in altering and maintaining the conductivities in the different regions of cGUVs (outer, annular, and inner) for a reasonable time. The conductivity of the inner region is set during the osmotic shock to sGUVs by modifying the salt concentration of the solution used for the osmotic shock; the inner vesicle engulfed the solution inside with the adjusted conductivity, thereby defining the inner region's conductivity. The conductivity of the annular region is determined by the conductivity of the hydrating solution used during the formation of the cGUV. The conductivity of the outer region can be modified after the cGUVs are formed. This is achieved by either adding a salt solution to the external medium or through dilution to reach the desired conductivity level in the outer region.

In prepared cGUVs, both deflated and well-formed quasi-spherical are observed. sGUV synthesis through electroformation typically exhibits high membrane tension, ranging from 10^-5 to 10^-7 mN/m, which promotes the formation of highly spherical vesicles. However, when these GUVs were subjected to an osmotic shock, the membrane tension decreased due to efflux of water from the inside of the vesicle, leading to deflation of the vesicles. Following the dissipation of the osmotic shock, a mixed population of cGUVs with both good sphericity and deflated vesicles was observed. Our previous work found that the membrane tension for cGUVs lies between 10^-7 and 10^-9 mN/m. The membrane tension in this range shows a reasonable agreement with theoretical models predicting the electrodeformation behavior of cGUVs, further supporting the validity of our experimental observations²¹. This also indicates that the cGUVs have lower membrane tension compared to sGUVs synthesized using the electroformation method. It was also observed that with time (after 6 h), a significant population of cGUVs attained sphericity, indicating recovery of membrane tension.

This method shows that a significant population of cGUVs can be synthesized, as shown in Figure 6, when the lipid composition is made of DMPC/Chol (63:37) with good reproducibility. In some cases, when temperature and osmotic shock conditions are not adequately controlled, the yield can vary. Future studies should focus on analyzing the role of cholesterol under different temperature conditions and its domain formation within the bilayer. Additionally, understanding how osmotic shock redistributes cholesterol and alters lipid packing, leading to increased bilayer fluidity and changes in bilayer mechanical properties, would provide valuable insights.

In the literature, fabricated microfluidic devices employing jet flows have been used to project liposomes onto planar lipid bilayers to achieve vesicle-in-vesicle geometry. However, this method is typically suitable for the entrapment of smaller-sized liposomes and an uncontrolled number of them within the vesicle³⁰. The method proposed in this work offers a convenient and straightforward approach for synthesizing compound vesicles (cGUVs) that contain a single micron-sized inner vesicle for conducting concise biophysical studies. It is particularly useful for experiments where spatial electrical conductivity variation and encapsulation of molecular dyes are required. Additionally, to extend the preparation technique, the electroformation of GUVs using the existing setup can be replaced with a portable GUV preparation kit. This kit includes a feature for heating electrodes as needed to reach 40 °C above the phase transition temperature of DMPC lipid, and is easy to operate^31,32.

However, some limitations must be addressed to develop an improved method for cGUV formation. The synthesis process is optimal at low salt concentrations or without salt. Since higher salt concentrations modify bilayer properties, preventing the GUV from transitioning into the VIV structure, difficulties are encountered in their synthesis. This method to synthesize cGUVs is time-consuming, as it takes hours to complete the process. Furthermore, this method is limited to specific lipid compositions; adding cholesterol introduces complexity, making it less suitable for studying GUV bilayers composed of a single lipid type. Therefore, a comprehensive investigation of other lipid compositions is essential to explore this method further for forming cGUV.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cholesterol (Sigma grade , 99%)	Avanti Polar Lipids, Inc.	C8667	For sGUV synthesis
Choloroform	Merck Life Science Pvt. Ltd.	DA2P711602	used for solvent for preparation of lipid stock solution
DMPC	Avanti Polar Lipids, Inc.	850345C	For sGUV synthesis
Ethanol	Hayman	F204325	For cleaning of ITO slides
FITC-DEXTRAN (4 Kda-MW)	Sigma-Aldrich	466994	For encapsulation inside the inner vesicles of cGUV
Glucose	Sigma-Aldrich	G-7021	For preparation of glucose solution, used for osmotic shock to sGUV suspended in the sucrose solution
Liss Rhod PE	Sigma-Aldrich	810150C-1MG	To stain a lipid molecules
Nile red	Sigma-Aldrich	72485	For staining of cGUV and sGUV membrane
Sodium chloride (NaCl)	Sigma-Aldrich	1936060521	For preparation of salt stock solution, to change the conductivity  of the cGUV compartments
Sucrose	Sigma-Aldrich	S9378	For sucrose sol. as a hydrating media of the electroformation chamber
Equipments
Cavitly slides	Blue star		cGUV suspension placed for microscopy observation
cMOS pco.edge 4.2 camera	Excelitas	61009524	For fluorescent monochromatic  images and DIC imaigng
Cover slip (22 x 60 mm)	Blue star		For covering of cavity slides
Eppendrof Electrofusion chamber	Sigma-Aldrich		sGUV suspension placed in the chamber for osmotic and microscopy observation, any other simple chamber can also be used
Function generator (Dual channel)	Tektronix	AFG3022C	Function generator used to deliver AC electric field to electroformation  chamber
Incubator	local customised		electroformation chamber placed at 40?
ITO slides	Sigma-Aldrich	576352-10PAK	ITO slides used in electroformation method
Nikon Eclipse inverted micrscope	Nikon	TE2000-U	For visulissation of sGUV and cGUV
Silicone sheet (3 mm  thickness)	Sigma-Aldrich	GF49463141	For spacing in electroformation chamber
Syringe (2 mL)	Becton Dickinson India Pvt Ltd.	300844	Used in injecting a hydrating media and harvesting the sGUV from electroformation chamber
Zeiss confocal microscopy setup	Central facility of IIT Bombay