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

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.

We've conducted an elaborate study on the synthesis and electro hydrodynamics of compound giant unilamellar vesicles to establish them as biomagnetic equivalents of eukaryotic cells. The attempt is in understanding technologies involving application of electric field to biological cells such as cell electroporation and cell electrodeformation.

A combination of a fluorescence and light microscopy, nanosecond pulse electric treatments, oscilloscope and power sources, and innovative synthesis methods are employed to advance the research in this area. The challenge in the synthesis of well-formed compound GUVs in the sensitivity of a method to a temperature, types of lipids, and the composition used. In this work, we demonstrate this for a DMPC and cholesterol system, but generalizing it remains challenging. Simple GUVs that mimic a nucleic cells have been synthesized in the literature. We synthesize the compound giant vesicle to emulate a nucleated cells with the valve defense structure, enclosing a inner vesicle of roughly half of the size of the outer vesicle.

We will focus on studying the effect of micro and nanosecond pulses on the inner vesicle, which is a mimic for the nucleus of a biological cell in the context of electroporation.

[Narrator] To begin, thoroughly clean the indium tin oxide or ITO-coated slides using a dish soap solution, and rinse with deionized water with a conductivity of 0.055 microsiemens per centimeter. Then using a 100% ethanol solution, clean the slides again and rinse with deionized water. Dry the slides in an oven set to 85 degrees Celsius. Identify the conductive side of each ITO-coated slide using a clamp meter. Secure the clean slide onto the spin coder stage using a vacuum, ensuring that the conductive side is facing upward. Apply 25 microliters of the lipid solution, one is four droplets onto the conductive side of one ITO slide. Take another slide and apply 25 microliters of the lipid solution two in the same manner. Vacuum dry both lipid coated slides in a desiccator stored in the dark for a minimum of two hours. Then arrange a lipid-coated ITO slide with lipid one in parallel with a clean slide, ensuring the conductive sides face each other, and place a three-millimeter thick silicone rubber spacer between them. Seal the setup with a clamp to create an electroformation chamber. Now fill the chamber with 100-millimolar sucrose solution using a two-milliliter syringe. Connect the output cable of the function generator to the ITO-coated slides with the function generator. Place both electroformation chambers in an incubator maintained between 38 and 40 degrees Celsius. Apply an alternating current electric field of five volts peak-to-peak at 10 hertz frequency using a dual-channel function generator for four hours. After incubation, disconnect the electroformation chambers from the function generator. Remove them from the incubator, and allow them to cool to room temperature. Using a syringe, harvest the synthesized giant unilamellar vesicles from each chamber and transfer them into separate two-milliliter microcentrifuge tubes. Incubate the tubes at room temperature between 25 and 27 degrees Celsius for one hour before proceeding to osmotic shock. Transfer 200 microliters of simple giant unilamellar vesicles, or sGUVs, suspended in hydrating media containing 100-millimolar sucrose solution into the observation chamber, and introduce 125 microliters of 300-millimolar glucose solution to induce osmotic shock and initiate shape transitions. Allow the sGUVs, now undergoing osmotic shock, to rest for one hour inside the observation chamber placed on the microscopy stage. Perform differential interference contrast and epifluorescence microscopy using an inverted microscope equipped with a monochromatic camera. Use Plan Fluor extra-long working distance 20X by 0.45 and 40X by 0.60 objective lenses to observe shape transitions in the sGUVs. For epifluorescence, use a green filter set with excitation at 510 to 560 nanometers, a dichroic mirror at 575 nanometers, and a barrier filter at 590 nanometers for Niall Red-stained bilayers. Monitor shape transitions within the observation chamber using differential interference contrast and epifluorescence microscopy with Plan Fluor objective lenses. Observe the formation of stomatocytic vesicles as the shape transition proceeds. Monitor how the larger vesicles settle first, followed by an increase in the number of smaller vesicles over time. Next, add salt solution to adjust conductivity in different regions of the cGUVs before inducing osmotic shock. For example, to create higher conductivity in the outer and inner regions than the annular region, transfer 200 microliters of sucrose solution into the electrofusion chamber. Add 20 microliters of 7.5-millimolar salt solution into the chamber, and induce osmotic shock with 125 microliters of 300-millimolar glucose solution. Allow the osmotic shock sGUVs to rest in the chamber for three to four hours. Confirm that the resulting cGUVs have higher conductivity in the outer and inner regions compared to the annular region. To perform electrodeformation of the cGUVs, space the wire electrodes 500 micrometers apart, and apply an alternating current electric potential of 7.5 volts peak-to-peak at 100 kilohertz using a function generator. After the electric field is applied, capture video at 10 frames per second, and observe the oblate deformation of the outer vesicles and the prolate deformation of the inner vesicles. Then load the cGUV mixture into a cavity slide and seal with a cover slip to prevent movement. Use a laser scanning confocal microscope to analyze cGUV morphology through Z-axis scanning with a step size of one micrometer. Employ a Plan Apochromat 40X by 1.3 oil DIC objective lens for imaging. Use a red single-channel mode with 561 nanometers excitation and 561 to 695 nanometers emission to image cGUV stained with Niall Red or Rhodamine PE. Modify and extract the .CZI image file using the microscope-linked software. Insert graphics like scale bars, and enable 2D and 3D views. Then go to processing method and parameters to adjust the desired settings. Then click Apply to export the image in JPG format. Finally, open the file in ImageJ software. To insert a scale bar, go to analyze, followed by set scale to calibrate, then select analyze followed by tools and scale bar to apply it. Confocal Z-stack imaging confirmed that inner vesicles were fully separated from the outer vesicles and elevated in the Z plane due to lower density of the inner solution. The intermediate stomatocyte state showed a narrow neck connecting inner and outer vesicles before complete separation. A diverse population of vesicular forms, including multiple inner vesicles, star-shaped bodies, and tubular structures were observed six hours after osmotic shock. A high abundance of cGUVs was formed using a lipid mixture of 1,2-Dimyristoyl-sn-glycero-3-phosphocholine and cholesterol in a 63 to 37 molar ratio. Under an alternating current electric field, cGUVs showed deformation with the outer vesicle forming an oblate shape and the inner vesicle forming a prolate shape.