Targeted Macromolecules Delivery by Large Lipidic Nanovesicles Electrofusion with Mammalian Cells

doi:10.4236/jbnb.2011.225063

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Journal of Biomaterials and Nanobiotechnology, 2011, 2, 527-532

doi:10.4236/jbnb.2011.225063 Published Online December 2011 (http://www.scirp.org/journal/jbnb)

527

Targeted Macromolecules Delivery by Large

Lipidic Nanovesicles Electrofusion with

Mammalian Cells

Pascal Demange1,2, Valérie Réat1,2, Stefan Weinandy1,2, Remy Ospital1,2, Louise Chopinet-Mayeux1,2,

Pauline Henri1,2, Alain Milon1,2, Justin Teissié1,2*

1Centre National de la Recherche Scientifique (CNRS), Institut de Pharmacologie et de Biologie Structurale (IPBS), Toulouse,

France; 2Université de Toulouse, Université Paul Sabatier (UPS), IPBS, Toulouse, France.

E-mail: *[email protected]

Received October 3rd, 2011; revised November 4th, 2011; accepted November 14th, 2011.

ABSTRACT

Lipidic nanovesicles (so called liposomes) were one the earliest forms of nanovectors. One of their limits was our lack

of knowledge on the delivery pathway of their content to the target cell cytoplasm. The present communication de-

scribes an efficient way to enhance the delivery. Pulsed electric fields (PEF) are known since the early 80’s to mediate

a fusogenic state of plasma membranes when applied to a cell suspension or a tissue. Polykaryons are detected when

PEF are applied on cells in contact during or after the pulses. Heterofusion can be obtained when a cell mixture is

pulsed. When lipidic nanovesicles, either small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs), are

electrostatically brought in contact with electropermeabilized cells by a salt bridge, their content is delivered into the

cytoplasm in electropermeabilized cells. The PEF parameters are selected to affect specifically the cells leaving the

vesicles unaffected. It is the electropermeabilized state of the cell membrane that is the trigger of the merging between

the plasma membrane and the lipid bilayer. The present investigation shows that the transfer of macromolecules can be

obtained; i.e. 20 kD dextrans can be easily transferred while a direct transfer does not take place under the same elec-

trical parameters. Cell viability was not affected by the treatment. As delivery is present only on electropermeabilized

cells, a targeting of the effect is obtained in the volume where the PEF parameters are over the critical value for elec-

tropermeabilization. A homogeneous cytoplasm labeling is observed under digitised videomicroscopy. The process is a

content and “membrane” mixing, following neither a kiss and run or an endocytotic pathway.

Keywords: Electrofusion, Delivery Systems in Cancer, Liposomes, Lipidic Nanovesicles

1. Introduction

New methods for large molecules delivery in cells re-

main a major problem in pharmacology. The main prob-

lem is to provide a pathway across the cell membrane

with no effect on the cell viability. Using liposomes (the

magic bullet) to target the delivery was a promising ap-

proach but it faces the limit of a poor transfer. Pulsed

electric fields (PEF) are known since the early 80’s to

mediate a fusogenic state of plasma membranes when

applied to a cell suspension or a tissue [1-3]. Polykaryons

are detected when PEF are applied on cells in contact

during or after the pulses [4]. Heterofusion can be ob-

tained when a cell mixture is pulsed. But the two partners

should have a similar size if the mixture is pulsed due to

the fact that the PEF effect is strongly size dependent.

Nevertheless we reported that when lipidic nanovesicles

(SUVs or LUVs) are electrostatically brought in contact

with electropermeabilized cells by a salt bridge, their

content was delivered in the cytoplasm [5]. This was

experimentally directly detected by the transfer of a

small soluble fluorescent dye, Pyranin, trapped in the

nanovesicles to the cell cytoplasm. A homogeneous cy-

toplasm labeling is observed under digitized videomi-

croscopy. The PEF parameters were adjusted to affect

specifically the cells leaving the vesicles unaffected by a

direct effect of the PEF. Under these specific experimen-

tal conditions, the nanovesicles were too small to be af-

fected by the field and thus only the electropermeabilized

state of the cell membrane was the trigger of the merging

between the plasma membrane and the lipid bilayer. No

Targeted Macromolecules Delivery by Large Lipidic Nanovesicles Electrofusion with Mammalian Cells

528

deleterious by-effect of PEF was detected on the cell

viability. The biophysical processes supporting the merg-

ing between intact nanovesicles and electropermeabilized

(fusogenic) cells is still under investigation. Membrane

fusion is a key process in life. It occurs when two mem-

branes, brought in close contact, merge in a single one. A

close approach of two membranes (as needed for fusion

to occur) is prevented by repulsive forces [6-8]. In the

case of synaptic vesicles, SNAREs bring the contact but

the following steps where a transient Ca2+ interfacial in-

crease occurs, are unclear [9,10]. The means, by which

the repulsive forces between the two partners are over-

come, remain poorly understood. But clearly a membrane

destabilization should occur [11]. It may result from

structural alterations of the target (plasma) membrane

under a transient electric stress [12].

Taking into account, on one hand that membranes are

electrically charged interfaces and on the other hand that

electrical modulations bring cell membranes to a destabi-

lized but spontaneously competent for fusion state, the

hypothesis of Rosenheck [13] that a rate limiting step in

membrane coalescence is due to the contact of electri-

cally destabilized membranes with small vesicles, can be

relevant of our observation. The model of Rosenheck

was recently extended to vesicles [14]. If electrofusion

between cells and lipid vesicles obeys the Rosenheck’s

model, then macromolecules transfer should be obtained

as in the case of exocytosis. This was checked in the

present study. Due to their large inner volume, large

unilamellar vesicles (LUV) were selected as nanovesicles

to give a significant delivery to the cytoplasm. Fluores-

cent dextrans were selected to monitor the resulting

transfer by digitized fluorescent microscopy and to valid

the proof of concept.

2. Materials and Methods

2.1. Cells

The wild type Toronto cells (WTT), is derived from

Chinese hamster ovary cells (CHOs) and was first intro-

duced in the 1960s. It can grow in suspension and in cul-

ture flasks (generation time = 18 - 20 hours).

The property to grow in suspension prevents the ne-

cessity of trypsinisation.

MEM 0111 buffer (Eurobio France (ref: CM1MEM-

40K-BP)) with Foetal Calf Serum 8%, (SVF EUROBIO,

ref: CVFSVF00-01, lot no: S155839), D (+) – Glucose

45%, (3.5 g/l) (Sigma, USA), Tryptose phosphate (2.95

g/l), Vitamins (GIBCO, ref: 043-01040) and Antibiotics

(penicillin 100 units/ml, streptomycin 100 mg/ml, L-

glutamin 0.58 mg/ml) is used to cultivate the CHO cells

under slow agitation (70 to 100 rpm, 37˚C). Cells stay in

the exponential growth phase by a control of the cell

number (dilution from 0.55 up to 0.7 × 106 cells every

day).

2.2. Vesicles

The lipids were Cholesterol (362794, SIGMA), L-α-

Phosphatidylserine (840032, AVANTI), 1,2-Dioleoyl-sn-

Glycero-3-Phosphocholine (DOPC, 850375, AVANTI).

8.5 mg of PC/PS/Cholesterol lipid mixture (6/1/3 mo-

lar ratio) was dissolved in chloroform. Lipids were dried

under nitrogen flow and vacuum (30 min). They were

then resuspended in 5 mM Hepes (pH 7.2) containing

Fluorescein labeled 20 kD dextrans (labeled-FD20) and

vortexed to form large multilamellar vesicles (LMV).

The temperature of HEPES buffer was kept above gel-

liquid crystal transition temperature of the lipid mixture

(i.e. −20˚C for DOPC). The suspension was sonified (3

times 5 minutes, with 3 min break between each treat-

ment) to obtain smaller unilamellar vesicles (SUV), then

centrifuged at 13,000 g to remove of the titanium debris

which came from the sonication tip. A 5 times freez-

ing/thawing process followed to form bigger vesicles.

Large unilamellar vesicles were obtained by extrusion

through 0.2 μm pore polycarbonate membrane (The

Mini-Extruder (AVANTI)). The size homogeneity was

checked by dynamic light scattering (Proteinsolution

Dynapro)

2.3. Electropulsation

Cells were suspended in a 10 mM HEPES buffer (pH

7.2), 250 mM sucrose and 5 mM CaCl2 at a high cell

density (4 × 107 c/ml). The “pulsing” buffer contained

CaCl2 to ensure the connection between cells and lipo-

somes via electrostatic interaction. Low conductivity was

important to decrease the Joule effect. A unipolar GHT

1287 generator (Jouan, France) was used to provide rec-

tangular pulses as high as 1 kV with a constant intensity

over the 100 μs pulse duration. An oscilloscope (Metrix

OX 520 B, France) ensured the monitoring of the pulse.

The generator was connected to two flat parallel stainless

steel electrodes with an inter-electrode distance of 4 mm.

The edges of the electrodes were in contact with the bot-

tom of a plastic Petri dish (Nunc 35 × 10) to build an

open pulsing chamber. Cells (0.1 ml at 4 × 107 c/ml)

were put between the two electrodes, together with a

labelled-Dextran (FD20) loaded LUV suspension and/or

HEPES buffer. 10 or more pulses (480 V = 1.2 kV/cm,

100 μs, frequency 1 Hz) were applied. After pulse deliv-

ery cells were kept 10 minutes at 25˚C.

A “washing” PBS buffer (Dulbecco’s phosphate buff-

ered saline, Eurobio, France, pH 7.2, -Ca2+, -Mg2+) was

added. It contained phosphate ions to chelate calcium and

remove the electrostatic bridges. The unfusioned nano-

vesicles were not bound anymore to the surface of the

Targeted Macromolecules Delivery by Large Lipidic Nanovesicles Electrofusion with Mammalian Cells529

cells and could be washed out by centrifugation (5 min,

100× g).

The cell pellet was resuspended once again in PBS and

analysed.

2.4. Analysis

Cells were analysed with the fluorescence digitized mi-

croscope Leica DM IRB (Wetzlar, Germany) using the

filter set for fluorescein. Video monitoring was possible

with a cooled CCD camera (Princeton Instruments, NJ,

USA). The pictures were taken with the Metavue soft-

ware (Molecular Devices, USA). Images were analysed

with ImageJ (for more information http://rsb.info.nih.gov

/ij/index.html). No filtering was operated on the raw data.

A ROI was selected and was analysed by the “surface

plot” operation.

3. Results

3.1. LUV Characterization

Fluorescein labeled 20 kD dextrans were used to make

sure that the electric treatment was not just inducing an

exchange (by a “kiss and run” process) of small mole-

cules as previously shown with Pyranin [5].

To eliminate the labeled 20 kD dextrans that was not

trapped in the vesicles, the extrusion method was fol-

lowed by a G200 chromatography. The elution profiles

are shown in Figure 1. LUVs were in the excluded frac-

tion volume (fraction 10 to 15). Strong fluorescein ab-

sorption was detected on a broad range of fractions,

starting from fraction 16. It was due to the elution of free

labeled 20 kD dextrans (untrapped in vesicles).

Furthermore, one single maximum in fluorescein ab-

sorption was detected suggesting homogeneity of the

fluorescent specie. This was indicative that no free fluo-

rescein (without dextran, i.e. with a low molecular

weight) was trapped in the LUVs.

LUVs were used after the gel chromatography.

Figure 1. Elution profiles at 400 and 480 nm Extruded LUV

solutions were analysed by gel chromatography. “Absorp-

tion” at 400 nm is due to the scattering of light by LUVs.

Absorption at 480 nm is due to fluorescein bound to dex-

trans.

3.2. Fusion

Labeled 20 kD dextrans could not be directly electro-

loaded in CHO cells under the electro pulsing conditions

of 10 × 0.1 ms [15].

The cells and LUVs (3/1, V/V) were premixed in

HEPES 10 mM, pH 7.2, sucrose 0.25 M, CaCl2 5 mM

(“pulsing” buffer). An electrostatic bridge bound the

negatively charged liposomes to the cells. No pulse was

applied. After 10 min incubation, the mixture was

washed in PBS buffer to chelate Ca2+ ions and remove

the free liposomes. Cells were then incubated at 37˚C up

to 4 hours. As shown in Figure 2, no evidence of fusion

was obtained as no fluorescence could be detected in the

cytoplasm. No endocytotic process was indeed detected

as no fluorescent spot could be detected even with the

very sensitive detection obtained with the cooled CCD

camera and the image processing. In order to get elec-

trofusion, a similar protocol was followed but 10 or 20

square pulses (1.2 kV/cm, 0.1 ms, 1 Hz) were applied on

the cell/LUVs mixture in the “pulsing” buffer. Fusion

was then detected by the content mixing making the cell

cytoplasm fluorescent as observed under a digitized

fluorescent microscope (Figure 3).

For both experiments, fluorescence was present in a

rather homogeneous pattern (an egg shell surface profile

was observed). The roughness of the distribution (Fig-

ures 3(a) and 3(b) surface plot) can be just relevant of

the noise of the signal as the magnitude of the “hot” spots

Contrast Fluorescence

Figure 2. No spontaneous fusion is present between cells

and LUVs Cells and LUVs were just mixed in the pulsing

buffer. No fusion with LUVs was observed without elec-

tropermeabilization as no fluorescence was detected in the

cytoplasm as shown on the right by the surface plot dia-

gram. The bar is 20 μm long.

Targeted Macromolecules Delivery by Large Lipidic Nanovesicles Electrofusion with Mammalian Cells

530

(a)

(b)

Figure 3. Cells after LUVs electrofusion observed under

fluorescence microscopy. (a) 10 pulses were applied. A gen-

eral view at moderate magnification, a zoomed view of a

single cell and its “surface plot” analysis are given; (b) 20

pulses were applied. A general view at moderate magnifica-

tion, a zoomed view of two “single” cells and their “surface

plot” analysis are given. Cells were observed 4 hours after

PEF delivery. The bar is 20 m long.

Is similar to what is observed in the background signal

detected outside of the cell. This may nevertheless sug-

gest that some endocytotic vesicles are present. The gen-

eral view at moderate magnification obtained on cells

treated by 20 pulses (Figure 3(b)) shows “hot” spots

very clearly (cells in the lower part of the figure). How-

ever, this observation is done with a wide field micro-

scope and the intensity of these “hot” spots is very high

(saturation of the camera) and so they may correspond to

stuck unfused-LUVs on the cell surface, rather than en-

docytotic vesicles. It is important to note that the ho-

mogenous cytoplasm labeling was observed only after 4

h incubation at 37˚C in complete culture medium (nano-

vesicles being washed out). No content mixing was ob-

served if cells were kept at 4˚C after PEF delivery. Fur-

thermore, more fluorescent cells in the pulsed population

are detected when increasing the number of pulses

(compared Figures 3(a) and 3(b)). There was however a

limit due to the loss of viability even if a protective effect

of the nanovesicles was present. Homogeneous FD entry

meant that the macromolecules were able to penetrate

within the nucleus. This organelle is rather large (see

Figure 2 the contrast picture to see its size) and should

give a poorly fluorescent area in the fluorescence image

if the macromolecules were not able to penetrate. This is

indeed not the case as displayed in Figure 3.

4. Conclusions

Fusion was obtained by using the electrical parameters

previously described to induce the cytoplasmic transfer

of the small sized Pyranin [5]. The homogeneity of the

cytoplasm fluorescence is due to its content mixing with

the internal volume of liposomes. Content mixing with

large molecules is the evidence of fusion of the nanove-

sicles, The process is a content and “membrane” mixing,

following neither a “kiss and run” nor an endocytotic

pathway [16-18]. The increase in fusion with the number

of pulses is linked to an increased fusogenicity with an

increase in electrically induced membrane defects. This

has been already observed in the fusion of cells with

Pyranin loaded vesicles [5] or in cell homofusion [19].

Content mixing for large molecules labeled 20 kD dex-

trans is obtained only after a long incubation suggesting

that a membrane-cytoskeleton reorganization must take

place.

Delivery by lipidic nanovesicles is associated to a tar-

geting and avoids the free circulation of the drug. The

lack of a direct electrotransfer of labeled 20 kD dextrans

was already reported [15]. LUV electrofusion can be

targeted either by a biological method (immunolipo-

somes) or simply by a targeted field delivery through a

proper localization of the electrodes.

Electrically enhanced binding of liposomes to cells

Targeted Macromolecules Delivery by Large Lipidic Nanovesicles Electrofusion with Mammalian Cells531

was reported 20 years ago [20]. The spontaneous desta-

bilization of the lipid bilayer of LUVs remains to be ex-

plained [21]. The field pulse induces a membrane desta-

bilization which is prone to a spontaneous fusion as pro-

posed by Rosenheck. Our results may be considered

within the framework of elucidating the physical basis of

exocytosis. The conclusion is that electropermeabiliza-

tion (and associated fusion) may be endogenously pre-

sent as already reported for peptide membrane permeabi-

lization [22]. A key feature of our delivery method is that

the transfer is obtained after a very short contact period

(10 min in this report), a major advantage for drug deliv-

ery. A final advantage is that the electrical conditions can

be used in clinical applications [23].

5. Acknowledgements

Supports from the region Midi Pyrénées, ANR Cemirbio,

PCV CNRS, ARC and Erasmus programs should be ac-

knowledged.

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