On the experimental setup for liposome

electroformation
Cite as: AIP Conference Proceedings 2302, 080007 (2020); https://doi.org/10.1063/5.0033867
Published Online: 03 December 2020

G. S. Valchev, L. S. Vladimirova-Mihaleva, and V. M. Vassilev

AIP Conference Proceedings 2302, 080007 (2020); https://doi.org/10.1063/5.0033867 2302, 080007

© 2020 Author(s).
 On The Experimental Setup for Liposome Electroformation
G. S. Valchev,1, 2, a) L. S. Vladimirova-Mihaleva,3, b) and V. M. Vassilev1, c)
1) Instituteof Mechanics, Bulgarian Academy of Sciences, Acad. G. Bonchev str., Bl. 4, 1113 Sofia, Bulgaria
2) Dept of Radiation Therapy, Queen Jovanna University Hospital, 8 Byalo more str., 1527 Sofia, Bulgaria
3) Faculty of Physics, St. Kliment Ohridski University of Sofia, 5 James Bourchier Blvd., 1164 Sofia, Bulgaria

a) Corresponding author: gvalchev@imbm.bas.bg

b) Electronic mail: vladimirova@phys.uni-sofia.bg
c) Electronic mail: vasilvas@imbm.bas.bg

Abstract. The cell membrane is a dynamic and complex barrier which separates the living cell from its environment. It consists
of a high variety of lipids and proteins and is continuously reorganized by the cell, making its in vivo study difficult. Thus one
has to use membrane model systems with precisely controlled composition to investigate fundamental interactions of membrane
components under well-defined conditions. Giant unilamellar vesicles (GUVs) are well-known model systems, especially because
they are easily observable using optical microscopy. In the present article we describe in details the experimental setup, applied
techniques and some preliminary results in the process of electroformation of giant unilamellar phospholipid vesicles (liposomes).
As a base of our studies we used the protocol reported by Pereno et al [1].

INTRODUCTION

Liposomes are nano- to microscopic spherical vesicles composed of single or multiple phospholipid (natural or syn-
thetic) bilayers enclosing a small volume of aqueous phase. Since the pioneering work by Bangham et al [2], lipo-
somes have become the prime model of biomembranes due to their biocompatibility, size and amphiphilic character.
The piling theoretical and experimental work during the last three decades led to the concept of the usage of liposomes
as delivery carriers for various bioactive materials [3, 4]. The amphiphilic nature of the compounds composing the
membrane of these vesicles allows them to encapsulate both hydrophilic agents into their inner aqueous core and
lipophilic ones within the membrane regions [5, 6]. Because the interactions between the phospholipid molecules
comprising the membrane are stronger than these with the inner aqueous medium, one observes elevated encapsula-
tion efficiency of lipophilic agents compared to hydrophilic ones. Among the biomaterials investigated for possible
encasement and subsequential transfer by liposomes are: anticancerous [7] and antimicrobial [8] agents, DNA and en-
zymes [9], vaccines [10], etc. The two principal parameters which determine the in vivo applications of liposomes are
their dimensions and distribution by size, as the first is essential particularly for drug loading, biodistribution within
the body, targeting efficacy, drug clearance rate and the overall therapeutic efficiency. Usually, liposome size ranges
from tens of nanometers up to several tens of micrometers, as the thickness of the lipid bilayer is approximately 4nm
[11]. For drug delivery applications, the desired liposome size vary between 50 and 150nm [12].
One can classify liposomes based on their lamellarity (number of bilayers) and size [13]. With respect to the first
attribute, one designates unilamellar (single bilayer separates the internal and external aqueous phase), multilamellar
(many bilayers (>5) entrap aqueous solution) and oligolamellar (small vesicles incorporated into a bigger one (∼0.1-
1μm)) liposomes [14]. According to their second characteristic, unilamellar vesicles (UV) are further divided into:
small (SUVs, 20–100nm), large (LUVs, >100nm) and giant (GUVs, >1μm). The most commonly used vesicle type
for incorporating hydrophilic molecules are LUVs, since they have comparatively larger captured volume (entrapped
volume of aqueous solution) than SUVs and multilamellar liposomes. On the contrary, the latter are a good choice for
incorporating hydrophobic molecules, since they have several lipid bilayer regions. GUVs are often used as membrane
model systems due to their larger size which enables visualization by optical microscopy and micromanipulation of
individual vesicle [15]. Moreover, GUVs are often employed for bottom-up strategies to mimic and investigate the
mechanical properties of cells. Such studies are of importance concerning cell’s exo- and endo-cytosis, adhesion,
growth and migration. The way cells respond to external deformation originates mainly from the plasma membrane
firmly attached to the contractile cytoskeleton, which is composed of cross-linked actin filaments as well as motor
proteins such as myosin II [16].
Natural swelling as an approaches to form GUVs was introduced by Reeves and Dowben in 1969 [17]. In their
original work they describe vesicles grow from prehydrated layers of stacked lipid bilayers due to a combination
of osmotic pressure, electrostatic interactions and the hydrophobic effect [18]. The lipids are initially dissolved in
chloroform and deposited on a solid substrate. As the solvent evaporates, the amphiphilic structure of lipids leads to
the clustering of several stacks of bilayers. By adding a buffer solution, vesicles can be obtained after several days.
Application of Mathematics in Technical and Natural Sciences
AIP Conf. Proc. 2302, 080007-1–080007-5; https://doi.org/10.1063/5.0033867
Published by AIP Publishing. 978-0-7354-4036-4/$30.00

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 In their pioneering work, Angelova and Dimitrov showed that the swelling process can be enhanced by introducing
a DC electric field [19]. In a later publication they describe an improvement of their method consisting in applying
an AC field which leads to a higher GUV yield due to the periodic redistribution of charges [20]. With the method of
electroformation, large amounts of cell-sized vesicles can be obtained within few hours in distilled water or sucrose.
As described, the lipids are deposited on two electrodes, and the swelling of the dried lipid film into vesicles is
enhanced by an electric field due to an interplay between electrostatic interaction, redistribution of bilayer counter
ions, membrane surface and line tension changes, as well as electro-osmotic effects [20]. Upon application of an
electric field, the lipids follow the alternating electric field depending on their net charge. The non-covalently bound
counter ions of the lipids redistribute between the bilayers following the electric field. Furthermore, the electric field
induces weak dipoles in the solution, causing a flow and membrane fluctuations, which lead to the separation of the
bilayers if the electro-osmotic effects overcome the van der Waals attraction between the bilayers [21]. In this case,
the water influx in between the bilayer stacks is increased and the separated membrane bulges out. The resulting
membrane buds are densely packed on the surface of the lipid layers. Due to the dense packing and the influence
of the electric field, the membrane buds fuse which can be observed during the formation process. By decreasing
the frequency of the AC field, the swollen vesicles follow the slow change of the field and detach from the surface,
forming free floating GUVs [21].
In this article we are going to describe in details the protocol used to prepare GUVs by the method of electroforma-
tion, following the work of Pereno et al [1].

MATERIALS AND METHODS

Materials

We used 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cholesterol (Ch), purchased from Sigma-Aldrich

(P6354 and C8667, respectively) (see Figure 1a), as components to form the lipid bilayer; D-(+)-sucrose, D-(+)-
glucose, chloroform, methanol, toluene and isopropanol were purchased from Valerus (Sofia, Bulgaria). All the
aqueous solution were prepared in deionized water (1 − 5μS). Ultra high purity argon gas was used as an inert
medium. The temperature in the laboratory was kept at 20◦ C.

Electroformation Protocol

We first prepare a mixture of chloroform and methanol in 2:1 v/v ratio, using 25ml and 50ml volumetric flasks,
washed with chloroform and dried under argon. Without removing the rubber sealing of the glass vessel containing
25mg DOPC we put a spinal needle (25G), with stylet removed, whose plastic hub is loosely connected to an argon
gas stream (see Figure 1b-2). We do this in order to equalise the pressure within the container and the atmospheric
one, as well as to minimize external impurities to enter the volume. After that, using 100μl glass syringe (see Figure
1b-1) we inject 300μl of the prepared mixture in the volume of the DOPC containing vessel (see Figure 1c). Prior
to this, not to influence the purity of the phospholipid, both needles were rinsed with isopropanol and dried under
argon. Finally we remove the rubber sealing and transfer the content of the glass vesicle into an amber reagent bottle
equipped with PTFE screw cap and seal, to reduce evaporation. Additionally the interior of the DOPC container is
washed several times to minimize loss of phospholipid. The bottle is then filled with enough solvent mixture to render
concentration of the DOPC in it of 1mg/ml (see Figure 1d). In an analogical manner we prepare cholesterol solution
with concentration 10 mg/ml: 500 mg of the substance (see Figure 1e) are weighted, using 4.5ml (19mm/9mm)
glass weighting bottle and electronic precision balance (PCB 250-3 - KERN & SOHN GmbH) (see Figure 1f), and
afterwards dissolved in 50ml of the prepared solvent mixture.
Following Ref. [1], we then prepared solutions of DOPC and cholesterol at varying molar ratios (1 : 0−1 : 3); using
adjustable-volume micropipettes equipped with plastic tips, pre-wetted in chloroform to prevent leakage of the intake
solutions. To prepare DOPC:Ch of 1 : 1 (see Figure 1g), 1 : 2 and 1 : 3 mol/mol ratios we added to 427μl DOPC
solution 21, 42 and 63μl Ch solution, respectively.

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FIGURE 1.

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 Afterwards, 50ml 0.2M sugar solutions was prepared, dissolving 3.423g sucrose and 1.982g glucose in 50ml deion-
ized water. The amount for each substance was measured using plastic weighting bottle (see Figure 1h-1) and an
electronic balance. The substances were then put in two separate 50ml glass beakers (see Figure 1h-4) and 30ml
deionized water was added. After complete dissolution each of the two contents was poured in volumetric flasks (see
Figure 1h-2), which were filled out (using PE wash bottle) up to the marker, in order to render the desired molar
concentration. The so prepared solutions are then transferred in gamma radiation sterilized self standing Falcon test
tubes (see Figure 1h-3), put in an desiccator (see Figure 1i-1) and degassed for 5h under reduced pressure of 100mbar,
achieved with a standard duty diaphragm pump (WelchTM MPC 090 E) (see Figure 1i-2).
The electroformation chamber (see Figure 1k) is composed of two parts: containing cell – single 4ml polystyrene
cuvette with outer dimensions 13 × 13 × 45mm and size of the opening of 11 × 11mm; pair of electrodes – two spinal
needles (25G (orange stylet hub); outer diameter 0.5mm) with removed stylets, fixed at a distance of 3mm between
their opposing surfaces through a couple of LDPE square caps. The separation was assessed via printing magnifying
glass, equipped with fine graduated metrical leaf-stand. To maintain the high purity of the experimental set up, prior
to the assembly of the chamber the interior of the cuvette was rinsed with isopropanol and dried under argon. The
electrodes together with the holding caps were immersed in toluene for 24h and wiped using Kimwipes (Kimberly-
Clark Professional). This was performed to remove any traces of lubricant from the needles surface and bio-organic
depositions on the fixation caps. In order to increase the contact surface, both electrodes are roughened by coarse sand
paper. Subsequently, both the caps and electrodes were washed with deionized water and dried under argon. This
intends to remove fibers and any small size particles, left by the wipes and the sand paper.
The desired solution (DOPC:Ch mol/mol in 2:1 choloroform:methanol) was then pipetted horizontally onto the
electrodes (5μl per electrode) using a 50μl glass syringe, carefully coating the entire surface. The pair was then put
in the desiccator and dried for 3h under reduced pressure of 100mbar (see Figure 1i).
After this phase the electroformation chamber was assembled in a vertical configuration (as shown on Figure 1k).
Two additional holes were bored on the sealing cap. The first one is situated at the vicinity of the cell side, located
at a maximum distance from both electrodes, and is used to fill the chamber volume with the sucrose solution. The
second (made between the electrodes) lets the contained argon gas flow out of the chamber during the filling process.
The actual filling is done through 0.2 or 0.45μm cellulose acetate membrane filter (as shown on Figure 1j-1), with
mounted, on one side, to a disposable sterile plastic syringe (filled with ∼ 5ml sucrose solution), and on the other –
to a spinal needle (22G (black stylet hub); outer diameter 0.7mm). Filling of the interior of the cell is done slowly
starting at the bottom of the chamber. To limit the turbulence of the aqueous medium, the tip of the needle has to be
maintained both close to the chamber side and the gas/aqueous medium interface during the fill.
The final step consist in connecting a signal generator (B&K Precision 4063) (see Figure 1l-2) via thin copper
wires to the electrodes of the chamber. A 5Vpp, 10Hz sinusoidal excitation was applied for 2h to induce repetitive
stress on the hydrated lipid bilayer, which has to lead to vesicle swelling. The effective input and output voltage
and frequency on the electroformation chamber were monitored using USB oscilloscope (PicoScope 2204A) (see
Figure 1l-3) connected to a PC (see Figure 1l-1). Subsequently, the frequency of the excitation was lowered to 5Hz
for 30min to facilitate vesicle detachment. At this point we observed a reading of 4.78Vpp, which indicated 0.22V
voltage difference, that might be related to occurrence of unmatched load of the signal generator. At the end of the
electroformation process, the electrodes, together with the fixation caps, were removed and the content was slowly
pipetted and divided equally into four 2ml Eppndorf snap-cap tubes, using a 100-1000μl pipette tip. To minimize
the shear stress at the orifice, the end of the pipette tip was cut by 2mm using a scalpel. The tubes were filled out with
additional 1 ml glucose solution up to the limiting volume of the tubes. This last step is to sediment and concentrate
at the bottom any formed sucrose containing lipid vesicles. The tubes were then stored in refrigerator for 24h before
ascertaining the result from the electroformation process.

PRELIMINARY RESULTS

In order to assess whether GUVs were formed, we slowly pipetted 50μl from the bottom of any of the storing tubes,
using 10-100μl pipette tip cut by 4mm. The drop is then placed on microscope slide glass, beforehand rinsed with
isopropanol and wiped with Kimwipes. We then carefully shake the glass until the formation of thin liquid film.
The observation is performed with binocular compound light microscope at 40× magnification. So far we have not
been able to observe any liposomes. Nevertheless we continue our studies using other imaging techniques as well as
employing different phospholipids and/or mixtures of such.

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 ACKNOWLEDGMENTS

The authors G.V. and V.V. gratefully acknowledges the financial support via Contract No. H22/2 of Bulgarian NSF.
L.V. acknowledges the financial support via Contract No. 777714 of H2020 RISE NOCTURNO.

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