R C Qiao

X Cheng

K X Shen

G Dourneau

S H Wang

X Y Hu

Z H Tang

X J Xi

Astrometric observations of Nereid in 2006-2007

Keywords:

 was used in the reduction so that a classical astrometric calibration method was applied. We have shown that our observations of Nereid appear to be of equal or higher precision (σ 0.2") than most of the recent CCD ones.

INTRODUCTION

Since Kuiper discovered Nereid in 1949, this satellite has been poorly observed due to its extremely difficult observing conditions: an extreme faintness, with a visual magnitude mv of about 19.5, and an irregular orbit. However, after the Voyager 2 spacecraft successfully accomplished a passage through the Neptune system in 1989 August 25, Nereid has attracted some more interest and attention of the scientific community. In order to facilitate the future space exploration, more knowledge of the positions of this small satellite has become a pressing requirement.

Nereid is one of the most distinctive satellites that we know in the solar system. The distance from Nereid to its Primary much varies, from about 1,4 to 9,6 million km, due to its very large excentricity (e 0.75). Furthermore, its orbit has a rather long period of about 360 days and a large inclination with respect to Neptune's equator (i 27 degree). These distinctive features of Nereid's orbit have made this satellite difficult to observe, hindering further research on it. Based on these peculiarities, the study of Nereid's orbit is

The data are available in electronic form as Supplementary Material to the online version of the paper on Blackwell Synergy, at the CDS via Anonymous FTP to cdsarc.u-stasbg.fr or via http://cdsweb.u-strasbg.fr/Abstract.html. † E-mail: rcqiao@ntsc.ac.cn one of the inspiring ways to acquire better knowledge of the solar system. Towards this end, more efforts have been made for obtaining new highly accurate observations of Nereid. However, before our observations made in 2006-2007, only less than 600 observed positions were obtained in the past. In 1989, the Voyager 2 spacecraft approach near the Neptunian system provided 83 unpublished positions determined by Jacobson et al.(1991). All the other observations of Nereid are ground-based observations. Before the Voyager 2 approach, 70 astrophotographic observations were accomplished. The first three of them were made by Kuiper (1949). Then, van Biesbroeck observed Nereid in a continuing program of solar system objects at Mac Donald Observatory from 1950to 1969(van Biesbroek, 1951, 1957;van Biesbroek et al., 1976). All the data so obtained was used by Rose (1974) to present a new orbit of this satellite. Then, 17 new observed positions were obtained by Veillet (1982) and by Veillet & Bois (1988) who also derived a new orbit of Nereid from the theory developped by Mignard (1975Mignard ( , 1981)). Landgraf (1988) observed 5 new positions and Schaefer & Schaefer (1988) made 4 new ones. The last astrophotographic observations were made by Veiga et al. (1996), with 8 new positions. Later on, Veiga et al. (1999) 175 of these recent CCD observed positions, as available on their web site.

We initiated in 2006 a new attempt to perform CCD astrometric observations of Nereid. This Nereid campaign of observation is part of a wider campaign of CCD observing faint planetary satellites, that we began in 1994 (Qiao et al., 1999;2006;2007). Since 2006, for observing Nereid, we have been using the two reflectors of National Observatory at Beijing. Thus, we used the 1m reflector, and especially the 2.16m diameter reflector for its stronger power to detect faint objects than the 1.56m reflector of the Sheshan Station, near Shanghai, that we used in our previous campaigns of observations. As a result, we present in this paper the 112 accurate CCD astrometric positions of Nereid, that we obtained during both of 2006 and 2007 Neptune oppositions. Thus, an analysis of these observations is made to evaluate their level of accuracy and to compare it to that of observations made by previous authors.

OBSERVATIONS, MEASUREMENT AND REDUCTION

Our observations

All our astrometric observations of Nereid were performed at Xinglong Station (IAU code number 327), near Beijing, which is an observing station of National Observatory, Chinese Academy of Sciences. Xinglong Station is located at E117.577 degree, N40.396 degree and H940.0 meters. We successively used the 1m and the 2.16m telescopes, both equipped with large CCD chips of 1340 × 1300 and 2080 × 2048 pixels, respectively. Accordingly to the focal length of each telescope (7.8m and 10m) and to the size of the pixels of each CCD chip, the available field was about 11 ×11 for both of these instruments. The complete specifications of the two instruments with their respective CCD chips are listed in Table 1. A total of 112 astrometric positions were so obtained in four successive observing periods, spanning from 2006 to 2007. For the first one, in August 2006, we obtained 15 positions with the 2.16m telescope (set 216a). In the second period of August 2007, we used the 1m telescope to obtain 17 new positions (set 100a). The third period was just a few days after in the same month, and we observed 34 new positions with the 2.16m telescope (set 216b). During the fourth and last period of observation of this campaign, in September 2007, we used the 1m telescope for obtaining 46 new positions of Nereid (set 100b).

The flat-field images were taken at dusk and dawn. The bias is taken at the beginning and at the end of the observation. The dark field images were taken at the end. No filter was used during our observations of Nereid. The exposure time was about 2 -10 minutes, depending on the weather conditions, elevation above horizon, and the setting of the different telescopes. In Fig. 1, two typical CCD images of Nereid taken at different times are presented. These are raw images, without any dark or flat field correction, showing the original map of the data. In the frames, Nereid is indicated by an arrowhead, and its motion relative to stars appears quite visible.

Measurement and data reduction

In previous astrometric reductions of satellite observations, calibrating the CCD frame was very difficult, in connection with the lack of reference stars, due to the limitation of the small size of CCD chips and to the low density of previous star catalogues. Consequently, authors had either to use secondary star catalogues or theoretical differential positions of satellites with respect to other satellites or to the Primary, as did Shen et al. (2001) for the Saturnian satellites. In the peculiar case of Nereid, the high difference of its magnitude with Neptune's could affect the determination of its relative positions. With the development of technology, large CCD chips began to be available to observe astrometric positions of planetary satellites, as we did in our latest campaigns. In addition, with the recent high density astrometric accurate catalogue UCAC2 (Zacharias et al., 2004) providing more than 48 million reference stars, enough number of reference stars can be found in the same CCD frame to perform a classical astrometric reduction. Consequently, in this work, we have been able to directly obtain the absolute positions of the satellite Nereid, and so, its relative positions with respect to Neptune did not need to be measured.

In our four observing periods, the average value of Full Width at Half-Maximum (FWHM) was calculated for determining the value of seeing. The range of seeing in Xinglong Station appears to be about 1.5 -3.5 arcsec, typically 2.5 arcsec. We use the powerful ASTROMETRICA software to determine the positions of the star center and satellite images. Astrometric Data Reduction command applies a twodimensional Gaussian fitting to every image and a term of second-degree polynomial to represent the background level. The centering error of Nereid is rather significant, with about 0.08 arcsec, because of the extreme faintness of this small satellite. Accordingly, we had to take rather long exposures to obtain the images of Nereid and so, the images of stars brighter than 16 magnitude could be saturated in the same CCD images. Such CCD frames with saturated images were eliminated. For example, during the 216a set, we obtained more than 50 CCD frames. Many of them were taken with a rather long time exposure of 900s, due to variable bad weather conditions. Finally, we only kept the 15 CCD frames without any saturated images.

Since the high-dense star catalogue UCAC2 (Zacharias et al., 2004) was used for our astrometric calibration of the CCD frames, there are about 8 to 16 UCAC2 reference stars available in each CCD images. This number is quite sufficient to let us use the six-constant model of reduction. So, we performed an accurate classical astrometric calibration method, without using any secondary star catalogue or any other indirect method which could have affected the accuracy of the derived observed positions of Nereid.

In Table 2, as an example, we list some of our observed positions of Nereid. To most observers with a consistent data format these data are topocentric and given in the ICRF J2000 system. The complete data can be obtained on the web site of the CDS at the following address: http://cdsweb.u-strasbg.fr/Abstract.html, or via Anonymous FTP to cdsarc.u-stasbg.fr.

ANALYSIS OF OBSERVATIONS

Comparison with theoretical positions

In the 70's of last century, Mignard (1975Mignard ( , 1981) ) set up a theory of the motion of Nereid including solar perturbations. Since 90's, Jacobson (1990Jacobson ( , 1991) ) successively proposed several further improvements of the theory of Nereid. The equations of motion and variation equations used in the Jacobson's dynamical model, quite suitable for the construction of outer planet satellite ephemerides, are identical to that previously given by Peters (1981). In a forthcoming paper, we plan to develop a new numerical theory of Nereid after fitting the additional materials including our own observations. In Jacobson's model, the perturbation forces of Nereid include the gravitational effects of the oblate primary, the perturbations of the barycenter of Neptune-Triton and other external effects. We have compared our observations of Nereid to ephemeris of this satellite available on the IMCCE web site and derived from the theory of Jacobson (1991).

For the planet Neptune, we have used the DE405 planetary ephemeris [START_REF] Standish | Highlights of astronomy[END_REF]. In Table 3, we present the values of mean residuals µ and standard deviations to the mean σ of the absolute positions of Nereid for each of the four successive sets of observations defined above in Section 2.1. N is the number of observed positions for each set of observations. As we can see in Table 3, the values of standard deviations σ of our observations are about 0.2 arcsec in right ascension as well as in declination. This value can be considered rather satisfactory as Nereid is a very faint satellite difficult to observe. Surprisingly, the 100a set, made with the smallest telescope that we used all along this observational campaign, appears to be the most accurate, with σ 0.16" in right ascension and σ 0.13" in declination, possibly due to the quite good weather conditions.

The high accuracy of our observations has been achieved certainly as we have used large CCD chips and the high accurate star catalogue UCAC2. Figure 2 displays the observed minus calculated residuals as a function of time for our observations. We can see that the absolute value of most residuals is lower than 0.3 arcsec. Also, no significant systematic positive or negative bias is observed on Fig. 2, accordingly to Table 3 presenting mean residuals always under 0.04", excepted in right ascension for the sets 100b and 216b. The first of these sets presents a slightly positive systematic mean residual (µ 0.08") when the second set presents a hardly higher negative residual (µ -0.11"). However, these mean residuals remain very low and show that the accuracy of the Nereid Jacobson's ephemeris appears to be about 0.1 arcsec.

Comparison of our observations with other recent CCD observations of Nereid

The internet web site of the IMCCE provides some recent CCD Nereid observations ranging from 1993 to 2007 and made in different observatories as Itajuba, Mauna Kea, Cerro Tololo, Kambah, Montcabre, Palomar and Lulin. All these observations were not published, excepted those made in Itajuba (Veiga et al., 1999). They only had been collected by the Minor Planet Center which communicated them to the IMCCE. They represent a total of 175 observed positions, spreading over the period from 2001 to 2007. We have analysed all these observed data that we have compared to the theory to obtain positional residuals by using exactly the same procedure we used above for our own observations. Then, it has been possible to compare the accuracy of all these recent CCD observations to ours.

In Table 4, we have listed the mean residuals µ and the standard deviations to the mean σ for each set of these recent CCD observations, as we did in Table 3 for our observations. So, the residuals of Nereid presented in Tables 3 and4 involve all the available recent observations of this satellite from 1993 to 2007, all obtained with the CCD techniques. These residual statistics can appear as a complement for the recent observations of Nereid to those previouly given by Jacobson (1991) for all the earlier observations of this satellite, made from 1949 to 1989. Now, we can compare the standard deviations to the mean σ given in Table 3 for our observations to those given in Table 4 for the other recent CCD observations. We can see that our observations, with σ values of about 0.2 arcsec, are more accurate than many other recent CCD observations which can present σ values between 0.3 and 0.6 arcsec, as those made in Kambah from 2004 to 2007. However, some other observations can present equal or slightly better standard deviations than ours, as those made in 2002 with the NEAT/Palomar instrument which is equipped with a CCD camera presenting a wider chip than ours. Also, the observations made in Itajuba with 3 different chips, appear to be very accurate, with σ values very close to those obtained for our 100a set of observations. Now, the analysis of the mean residuals µ given in Table 4 for each set of observations shows that they generally are correlated with the values of the corresponding standard deviations σ. Consequently, as the most accurate sets of observations give rather low mean residuals, generaly hardly higher than 0.1 arcsec, we can confirm what we had shown above from the analysis of our own observations, that the accuracy of the used ephemeris of Nereid by Jacobson is about 0.1 arcsec. CONCLUSION In this paper, we have presented the 112 new CCD astrometric observed positions of Nereid that we have made in 2006 and 2007 at the Xinglong Station of Beijing observatory. Our observed positions present a rather high accuracy, with values of standard deviations to the mean of about 0.2 arcsec. This result should be due to the use of two large telescopes of 1m and 2.16m, equipped with large size CCD chips of 1340 × 1300 and 2048 × 2048 pixels. Also, the use of the high-density and high-accuracy star catalogue UCAC2 (Zacharias, 2004) in our astrometric reduction can have brought a supplement of quality in the resulting positions. Moreover, an analysis of other recent CCD observations of Nereid, available on the IMCCE web site, has shown that the accuracy of our observations is equal or better than most of them, even those which were made in the same years than ours (2006 and 2007).

So, we expect that the data presented in this paper will be of significant value for any future improvement of the knowledge of Nereid, and especially for our own research on this satellite, for which we plan to determine in a new study of the orbital parameters.

Figure 1 .

 1 Figure 1. Two typical CCD images of Nereid are presented. In the frames, Nereid is indicated by an arrowhead and its motion relative to stars is very clear.

Figure 2 .

 2 Figure 2. O-C of Nereid in 2006-2007 relative to the four sets of observations 216a, 216b, 100a and 100b from above to below

Table 1 .

 1 provided 230 new positions which were the first CCD observations of Nereid. More recently, new CCD observations of Nereid have been collected by the Minor Planet Center. The IMCCE presents Specifications of the two telescopes and CCD chips used for the observations of Nereid

		Telescope A	Telescope B
	Diameter of primary mirror	216cm	100cm
	Focal length	10000mm	7800mm
	Size of CCD array	2080x2048	1340x1300
	Size of pixel	15µm	20µm
	Angular extent per pixel	0.31	0.53
	Field of view	10.7 ×10.5	11.7 ×11.4

Table 2 .

 2 An extract of the list of the observed positions of Nereid

	year Month Day(UTC)	α	δ
	2007	08	08.692362 21 33 00.238 -14 49 27.99
	2007	08	08.702084 21 33 00.184 -14 49 28.59
	2007	08	08.711807 21 33 00.121 -14 49 28.79
	2007	08	09.564583 21 32 54.742 -14 49 55.50
	2007	08	09.569444 21 32 54.691 -14 49 55.37
	2007	08	09.574306 21 32 54.670 -14 49 55.73

Table 3 .

 3 Values of mean residuals µ and standard deviations to the mean σ relative to the four successive sets of observations

	set		N	σ(")	µ(")
	216a (2006/08)	∆αcosδ	15	0.215	0.001
		∆δ	15	0.235	-0.036
	216b (2007/08)	∆αcosδ	34	0.194	-0.112
		∆δ	34	0.191	0.026
	100a (2007/08)	∆αcosδ	17	0.161	0.009
		∆δ	17	0.126	0.020
	100b (2007/09)	∆αcosδ	46	0.200	0.082
		∆δ	46	0.193	-0.035

Table 4 .

 4 Mean residuals µ and standard deviations to the mean σ of other CCD observations of Nereid

	Set	year	N	µ	µ	σ	σ	Observatory
				(∆αcosδ)	(∆δ)	(∆αcosδ)	(∆δ)
	1	1993-1998	230	0.027	-0.130	0.184	0.150	874 -Itajuba (Laboratorio Nacional de Astrofisica)
	2	2001	3	-0.070	0.333	0.153	0.008	568 -Mauna Kea
	3	2002	3	-0.286	0.077	0.133	0.160	807 -Cerro Tololo Observatory, La Serena
	4	2002	11	-0.131	0.178	0.125	0.101	644 -Palomar Mountain/NEAT
	5	2003	1	-0.389	0.283			568 -Mauna Kea
	6	2004	7	0.533	0.082	0.588	0.392	415 -Kambah, near Canberra
	7	2006	18	-0.036	0.182	0.210	0.193	415 -Kambah, near Canberra
	8	2006	3	0.060	0.615	0.117	0.247	213 -Observatorio Montcabre
	9	2006	72	0.037	0.027	0.344	0.282	415 -Kambah, near Canberra
	10	2006	1	0.057	-0.705			D35 -Lulin Observatory
	11	2007	11	-0.271	0.067	0.290	0.350	415 -Kambah, near Canberra
	12	2007	45	0.056	-0.156	0.270	0.321	415 -Kambah, near Canberra

c 2008 RAS

c 2008 RAS, MNRAS 000, 1-5

ACKNOWLEDGMENTS

The authors are very grateful to the staff at Xinglong Station of the National Observatory for their assistance and especially to Dr. X. J. Jiang for providing us many conveniences throughout our observing run. This work was carried out with financial support from National Science Foundation of China (NSFC) (Grant Nos 10573018, 10333050, 10673026) and 863 program (Grant No 2006 AA12 Z322).


Angela Tesse

Franck M André

Thierry Ragot

Aluminum particles generated during millisecond electric pulse application enhance adenovirus-mediated gene transfer in L929 cells

Keywords: electrodes, aluminum particles, endocytosis, L929

published or not. The documents may come

Aluminum particles generated during millisecond electric pulse application enhance adenovirus-mediated gene transfer in L929 cells

Introduction

Delivery of macromolecules or nano-objects with bioactive properties into live cells either grown in vitro or constitutive of living tissues is a fundamental and irreplaceable procedure in many fields of biotechnology and more and more in various fields of therapeutics.

Electrotransfer is one major tool for this aim. It intends to use external electric pulses to induce or facilitate the penetration of bioactive agents into target cells. So far, electrotransfer has been mainly used for intracellular delivery of plasmid DNA in a large range of cells and tissues. Moreover, diversification of pulse types and their combination, associated to a variety of devices for pulse delivery, have allowed to extend the scope of gene electrotransfer applications [START_REF] Mir | Electroporation-based gene therapy: recent evolution in the mechanism description and technology developments[END_REF][START_REF] Young | Electroporation-mediated gene delivery in Nonviral Vectors for Gene Therapy -Physical Methods and Medical Translation[END_REF]. Electrogenetherapy strategies like DNA vaccination and/or cytokine-based immunotherapy to stimulate anti-tumor immunity are promising strategies to fight cancer [START_REF] Lambricht | Clinical potential of electroporation for gene therapy and DNA vaccine delivery[END_REF], as suggested in several phase I/II clinical trials using DNA electrotransfer [START_REF] Spanggaard | Gene electrotransfer of plasmid antiangiogenic metargidin peptide (AMEP) in disseminated melanoma: safety and efficacy results of a phase I first-in-man study[END_REF][START_REF] Heller | Gene electrotransfer clinical trials[END_REF]. Our knowledge on electrotransfer has been obtained both through theoretical and empirical approaches, but, while it is already used for wide industrial and medical applications, there are still major uncertainties and sustained controversies about the molecular and supramolecular effects of electrical pulses allowing the plasma membrane crossing by bioactive agents [START_REF] Young | Electroporation-mediated gene delivery in Nonviral Vectors for Gene Therapy -Physical Methods and Medical Translation[END_REF][START_REF] Satkauskas | Towards the mechanisms for efficient gene transfer into cells and tissues by means of cell electroporation[END_REF][START_REF] Teissié | Electrically mediated gene delivery : basic and translational concepts in Novel Gene Therapy Approaches[END_REF][START_REF] Pavlin | New Insights into the mechanisms of gene electrotransfer -Experimental and theoretical analysis[END_REF][START_REF] Venslauskas | Mechanisms of transfer of bioactive molecules through the cell membrane by electroporation[END_REF][START_REF] Rosazza | Gene electrotransfer: a mechanistic perspective[END_REF].

It is more and more evident that electrotransfer mechanisms are dependent on the size of the penetrating object. The concept of hydrophilic pore formation induced by electric pulses in the membrane bilayer (-electroporation‖ phenomena) is probably relevant to the penetration of small molecules (up to a few nanometers), although we lack precise information on the nature and size of these pores [START_REF] Silve | Cell electropermeabilization and small molecules cellular uptake: The electrochemotherapy concept in Electroporation in Science and Medicine[END_REF][START_REF] Saulis | Size of the pores created by an electric pulse: microsecond vs millisecond pulses[END_REF][START_REF] Silve | Demonstration of cell membrane permeabilization to medium-sized molecules caused by a single 10 ns electric pulse[END_REF]. Studies trying to visualize the reality of electropores, predicted by molecular dynamics computations, are scarce and controversial [START_REF] Chang | Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy[END_REF][START_REF] Sengel | Imaging the dynamics of individual electropores[END_REF]. The concept of pore is clearly insufficient when dealing with penetration of large molecules, especially DNA, and a more complex course of events appears to have more relevance [START_REF] Breton | Investigation of the chemical mechanisms involved in the electropulsation of membranes at the molecular level[END_REF][START_REF] Kotnik | Membrane electroporation and electropermeabilization: Mechanisms and models[END_REF]. In this scenario, during the pulses, electropores appear in the plasma membrane, but more significantly, lipids are then oxidized in larger areas around generated pores [START_REF] Breton | Investigation of the chemical mechanisms involved in the electropulsation of membranes at the molecular level[END_REF]. The penetration of macromolecules seems to occur through these oxidized areas in a second step called -electropermeabilization‖, which lasts much longer (up to several minutes) after electric pulse application, until the plasma membrane resealing. Several studies have actually shown that DNA electrotransfer was a multistep process consisting of 1) cell membrane electropermeabilization, 2) DNA electrophoretic drift towards electropermeabilized sites, 3) DNA-membrane interactions, 4) translocation of the DNA across the membrane few minutes after the pulses [START_REF] Golzio | Direct visualization at the single-cell level of electrically mediated gene delivery[END_REF][START_REF] Faurie | Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation[END_REF]. This model is supported by studying in vitro the roles of cell electropermeabilization and DNA electrophoretic drift with, respectively, high voltage (HV) and low voltage (LV) pulses [START_REF] Kandušer | Mechanisms involved in gene electrotransfer using high-and low-voltage pulses -An in vitro study[END_REF][START_REF] Čepurnienė | Influence of plasmid concentration on DNA electrotransfer in vitro using high-voltage and lowvoltage pulses[END_REF][START_REF] Pavlin | The role of electrophoresis in gene electrotransfer[END_REF][START_REF] Haberl | Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level[END_REF][START_REF] Liew | Robust, efficient, and practical electrogene transfer method for human mesenchymal stem cells using square electric pulses[END_REF], and also by earlier observations made on gene transfer in vivo [START_REF] Mir | High-efficiency gene transfer into skeletal muscle mediated by electric pulses[END_REF][START_REF] Satkauskas | Electrophoretic component of electric pulses determines the efficacy of in vivo DNA electrotransfer[END_REF][START_REF] André | Efficiency of high-and low-voltage pulse combinations for gene electrotransfer in muscle, liver, tumor, and skin[END_REF]. The real nature of the translocation step is still unknown yet. Some authors have noticed that electric fields could stimulate the uptake of various macromolecules by electro-endocytosis [START_REF] Rols | Long-lived macropinocytosis takes place in electropermeabilized mammalian cells[END_REF][START_REF] Rosenberg | Incorporation of macromolecules into cells and vesicles by low electric fields: induction of endocytotic-like processes[END_REF][START_REF] Antov | Electroendocytosis: exposure of cells to pulsed low electric fields enhances adsorption and uptake of macromolecules[END_REF], including plasmid DNA [START_REF] Rosazza | The actine cytoskeleton has an active role in the electrotransfer of plasmid DNA in mammalian cells[END_REF]. But, while many publications seem to confirm the involvement of endocytosis in the translocation step [START_REF] Escoffre | Electromediated formation of DNA complexes with cell membranes and its consequences for gene delivery[END_REF][START_REF] Rosazza | Cholesterol implications in plasmid DNA electrotransfer: evidence for the involvement of endocytotic pathways[END_REF][START_REF] Wu | Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells[END_REF][START_REF] Chang | Role of specific endocytic pathways in electrotransfection of cells[END_REF][START_REF] Markelc | Inhibitor of endocytosis impairs gene electrotransfer to mouse muscle in vivo[END_REF][START_REF] Mao | Involvement of Rac1-dependent macropinocytosis pathway in plasmid DNA delivery by electrotransfection[END_REF][START_REF] Cervia | Distinct effect of endosomal escape and inhibition of endosomal trafficking on gene delivery via electrotransfection[END_REF][START_REF] Rosazza | Endocytosis and endosomal trafficking of DNA after gene electrotransfer in vitro[END_REF][START_REF] Wang | Ultrastructural analysis of vesicular transport in electrotransfection[END_REF], controversial reports evidenced DNA entry by a non-endocytic translocation through the permeabilized membrane [START_REF] Golzio | Direct visualization at the single-cell level of electrically mediated gene delivery[END_REF][START_REF] Faurie | Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation[END_REF][START_REF] Satkauskas | Electrophoretic component of electric pulses determines the efficacy of in vivo DNA electrotransfer[END_REF][START_REF] Pavlin | The role of electrically stimulated endocytosis in gene electrotransfer[END_REF][START_REF] Phez | How transient alterations of organelles in mammalian cells submitted to electric field may explain some aspects of gene electroporation process[END_REF][START_REF] Lesueur | Overcoming the specific toxicity of large plasmid electrotransfer in primary cells in vitro[END_REF].

Several theoretical models, more or less based on experimental results, were proposed to explain the -translocation step‖ of DNA electrotransfer, but no definitive reliable mechanism has been described so far.

Interestingly, there are several reports where electrotransfer is used for biomolecules distinct from nucleic acids, like proteins (β-galactosidase, bovine serum albumin or full antibody), or dextran [START_REF] Rols | Long-lived macropinocytosis takes place in electropermeabilized mammalian cells[END_REF][START_REF] Rosenberg | Incorporation of macromolecules into cells and vesicles by low electric fields: induction of endocytotic-like processes[END_REF][START_REF] Antov | Electroendocytosis: exposure of cells to pulsed low electric fields enhances adsorption and uptake of macromolecules[END_REF][START_REF] Bobinnec | Centriole disassembly in vivo and its effect on centrosome structure and function in vertebrate cells[END_REF]. However, to our knowledge, there is no report on the use of electrotransfer as a tool to induce or facilitate the intracellular penetration of viral particles. The first aim of the present study was to determine whether it was possible to extend the application of electrotransfer to nano-objects of such large size, by testing different electric pulse types. As a model system, we chose to use as a penetrating agent a recombinant adenovirus (rAd) expressing a reporter gene, and as a target a murine cell line, L929, which is found weakly transduced by human adenovirus type 5 (Ad5) [START_REF] Jullienne | Efficient delivery of angiostatin K1-5 into tumors following insertion of an NGR peptide into adenovirus capsid[END_REF]. Human Ad5-based vectors have been widely studied in molecular biology and used in preclinical research and in gene therapy protocols. The fight against COVID-19 pandemic has hastened its deployment as a recombinant virus-vectored vaccine in the regular population. Ad5 does not replicate efficiently in most rodent cells, however, murine as well as human tissues can be transduced with Ad5-based vectors which efficiently express their carried transgene [START_REF] Ragot | Efficient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdx mice[END_REF]. Ad5 initiates its cell entry by interaction between the fiber knob and the Coxsackie virus and Adenovirus Receptor (CAR), an adhesion molecule of the human host [START_REF] Bergelson | Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5[END_REF], with its murine homolog, mCAR, found in the mouse [START_REF] Bergelson | The murine CAR homolog is a receptor for coxsackie B viruses and adenoviruses[END_REF]. Once bound at the cell surface, Ad5 is taken up into clathrin-coated pits by receptor-mediated endocytosis [START_REF] Stasiak | Human adenovirus binding to host cell receptors: a structural view[END_REF][START_REF] Greber | Adenovirus entry: from infection to immunity[END_REF].

When CAR gene is weakly or not expressed, especially in some cancer cells [START_REF] Hensen | Adenovirus receptor expression in cancer and its multifaceted role in oncolytic adenovirus therapy[END_REF], Ad5 can binds cells by using other proteins of the host (in particular integrins α 3 β 1 and α v β 5 ) but with a lower efficacy [START_REF] Lyle | Integrin αvβ5 is a primary receptor for adenovirus in CARnegative cells[END_REF]. Therefore, virus particles cannot be considered as classical nonpermeant molecules for which cell electropulsation facilitates their direct penetration into the cytosol [START_REF] Silve | Cell electropermeabilization and small molecules cellular uptake: The electrochemotherapy concept in Electroporation in Science and Medicine[END_REF]. Furthermore, in contrast to naked DNA, once it has crossed the plasma membrane, Ad5 avoids the multiple impediments that may hinder its gene expression. In addition, there is no topological variation of structure that can influence virus entry and expression unlike plasmid DNA [START_REF] Escoffre | New insights in the gene electrotransfer process: evidence for the involvement of the plasmid DNA topology[END_REF]. Therefore, in a given cell type, we can directly estimate the virus penetration into cells by measuring the expression of the reporter gene, inserted into the viral genome.

In the event of positive results, our next objective was to explore the mechanisms of viral penetration induced by electric pulses. The expected benefits of this work are of two kinds:

1) in terms of basic knowledge, to get more insights about electric pulse impact on the interactions between targeted cells and large penetrating agents; 2) in terms of practical applications, to open new ways for virus-based gene therapies applied for example to target tissues with weak or no expression of virus receptors (particularly in tumor or muscle); or to promote efficient entry of mutated (detargeted) recombinant viruses into cells to be treated for therapeutic purposes.

Materials and methods

Cell Culture.

L-929 (ATCC-CCL-1), a subclone of murine strain L fibroblastic cell line and 293 (ATCC-CRL-1573), a human epithelial cell line isolated from human embryonic kidney and containing left end of Adenovirus 5 DNA (LGC Standards, Molsheim, France), were grown in DMEM medium + GlutaMAX with 1% non-essential amino acids (Gibco, ThermoFisher Scientific, Watham MA, USA) and 10% FBS (Invitrogen, ThermoFisher Scientific). CHO-hCAR was kindly provided by J.M. Bergelson; this epithelial-like cell line derives from a subclone of Chinese Hamster Ovary cell line, CHO-K1 (ATCC-CCL-61), stably transformed with a plasmid coding human CAR [START_REF] Bergelson | Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5[END_REF]. It was grown in F-12 HAM (Gibco) with 10% FBS. G418 sulfate (geneticin, Gibco) was added at a final concentration of 250 µg/ml to maintain selective pressure on transformed cells. FBS had been previously heated (30 min, 56 °C) to inactivate complement and filtered on a 0.22 µm filter to remove protein aggregates. Before further processing, 60-70%-confluent cells were detached from dishes with TrypLE Express solution (Life Technologies, Courtabeuf, France), centrifuged (235×g, 10 min, 4 °C), counted, and suspended in S-MEM (Gibco), which was previously incubated in a CO 2incubator (Heracell 240i, ThermoFischer Scientific), to maintain its pH around 7.

Adenovirus preparation, titration and cell infection.

A rAd, Ad-CMV-eGFP, expressing the enhanced Green Fluorescent Protein (GFP) under the control of the cytomegalovirus (CMV) early promoter was constructed and the virus stocks were purified using standard procedures as described [START_REF] Ragot | Adenoviral gene delivery in Methods in muscle biology[END_REF]. Briefly, the rAd was amplified on 293 cells that were harvested around 35 h after infection, disrupted by 5 cycles of freezing/thawing and cell debris were removed by centrifugation (2000×g, 10 min, 4 °C).

The supernatant containing the crude viral solution was purified by two successive CsCl equilibrium density gradients (90,000×g, 2 h and 100,000×g, 18 h, respectively) and CsCl was removed by gel filtration on a Sephadex G-25 PD-10 column (Cytiva Europe, Velizy-Villacoublay, France). Virus aliquots were stored at -80 °C in the conservation medium (10 mM Tris/HCl pH 7.5, 10 % glycerol). Viral solution was assayed on 293 cell monolayers for plaque forming units (pfu) and titrated by HPLC [START_REF] Blanche | An improved anion-exchange HPLC method for the detection and purification of adenoviral particles[END_REF] to estimate viral particles (vp) number. Vp/pfu ratio around 20 was obtained, in agreement with published values [START_REF] Mittereder | Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy[END_REF].

Potential alterations that would be caused by electric pulses on rAd were also tested by end-point dilution assays on 293 cells, before and after electric pulse application on viral suspension: no appreciable variations were found by comparing both results.

The NanoSight LM10 nanoparticle characterization system (Malvern Instruments Ltd, Malvern WR, UK), equipped with a blue laser (405 nm) illumination, was used for adenovirus characterization (particle size and distribution) after stock batch thawing. A mean capsid diameter of 110 nm was found in accordance with the literature. The small aggregates (of 2-3 particles) represented less than 10% of the viral particles. Adenovirus surface charge was also measured, using a Zeta potentiometer (Nanobrook 90PLUS PALS, Brookhaven Instruments Corporation, Holtsville NY, USA) equipped with a 35 mW red diode laser, 640 nm wavelength. Ad particles were diluted in PBS (pH 7) and Zeta potential was measured at 25 °C. A mean value of -20.95 mV (n=5, SEM=1.79) was found, indicating a low charge of adenovirus at physiological pH. The rAd was diluted in cold S-MEM without FBS (viral suspension) and added to cells, held on ice, either before or after electric treatment, at the multiplicity of infection (MOI in vp/cell) indicated. Uninfected controls contained similar dilutions of the virus conservation medium alone.

Electric pulse delivery and treatment protocols.

Microsecond high voltage electric pulse train (HV: 8 x 100 µs, 1 Hz, 130 kVm -1 ) and millisecond low voltage electric pulse train (LV: 10 x 20 ms, 10 Hz, 10 kVm -1 , unless otherwise specified) were delivered by a Cliniporator device (IGEA, Carpi, Italy). A 10 dBattenuator was connected inline after the Cliniporator to deliver LV pulses below 12 kVm -1 .

Experiments incorporating bipolar pulses or unipolar pulses with alternate polarities were conducted using an Electro cell B10 electropulser (βTech, Saint-Orens de Gameville, France). LV pulses delivered had the same parameters as those used for unipolar pulses, except inversion of polarity after 10 ms during the 20 ms-bipolar pulses, and after each pulse for pulses with alternate polarities. Schematics of electric pulses are shown in Supplementary Figure S1.

For pulse delivery, 5x10 6 cells/ml in suspension in 50 µl of S-MEM without FBS, held 15 min on ice, were put in a standard cuvette with 1 mm-spaced plate aluminum electrodes (Cell Projects, Arrietsham, UK). Non-pulsed controls were held in a cuvette in the same conditions but without pulse delivery. Except where indicated, cells were maintained on ice between 20 to 40 min after treatment; then, they were collected and diluted 200 times in complete medium at room temperature, distributed in 6-well plates in two technical replicates per experimental point, and incubated at 37 °C.

To determine the percentage of electropermeabilized cells, the non-permeant YO-PRO-1 (trimethyl-[3-[4-[(Z)-(3-methyl-1,3-benzoxazol-2-ylidene)methyl]quinolin-1-ium-1-yl]propyl]az anium; diiodide) dye (LifeTechnologies, Carlsbad CA, USA) was added (1 µM), just before the application of pulses and cells, kept on ice, were examined 40 min later by flow cytometry analysis.

Gene expression analysis.

The GFP gene expression was analyzed according to two parameters: the acquisition of GFP expression by target cells -in other words the conversion rate to GFP-positive (GFP + ) cells, in % of GFP + cells, and the GFP gene expression intensity -median of cell fluorescence intensity, in Relative Fluorescent Units (RFU). Cells were analyzed 24 to 72 h after the rAd-mediated gene transfer (AMGT) by flow cytometry. Treated cells (in duplicates) from 6-well plates were washed with 500 µl of PBS, trypsinized with 500 µl of TrypLE™ Express for 5 min at 37 °C, and wells were finally washed with 500 µl of complete medium. Cells were suspended and immediately analyzed by the flow cytometer (BD Accuri C6, Becton Dickinson, Franklin Lakes NJ, USA). GFP fluorescence was determined by setting the excitation at 488 nm, the emission at 530/30 nm with a BP filter, and recording 10,000 events per sample. GFP fluorescence parameters were analyzed using the BD Accuri CFlow Plus software. The gating strategy for flow cytometry is described in Supplementary Figure S2.

The GFP cell conversion with time was observed using a real-time live cell imaging apparatus (IncuCyte Zoom, Essen Bioscience, Ann Arbor MI, USA) to determine GFP expression kinetics from 2 up to 35 h after the AMGT. Cells were plated immediately after the electric pulse delivery into 6-well plates (in technical duplicates) at densities which were adjusted to keep subconfluence during the experiment. The dishes were left in incubator at 37 °C during 2 h to allow cells to settle at the bottom of the wells before introducing them into the imaging apparatus. The mean percentage of GFP-expressing cells was calculated from 18 (2 x 9) fields distributed into duplicated dishes and imaged every 3 h (see representative pictures taken by the imaging apparatus camera in supplementary Figure S3). Cell eccentricity (equals to 0 for round cells and tends toward 1 for elliptical fully adherent cells) was also calculated at each time point, by the IncuCyte Zoom software.

Aggregate observation, characterization, quantification, and experimental protocols.

Aliquots (100 µl) of various solutions with a neutral pH (S-MEM culture medium; Phosphate Buffered Saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 ;

Hepes-NaCl: 25 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid, 130 mM NaCl) were pulsed with low voltage pulses (20 x LV), or left unpulsed. After mixing with 4 ml of sterile water in 6-cm dishes, they were observed by optical microscopy (Evos XL Core, ThermoFischer Scientific), using a phase contrast 20x-objective. The protocol used for the determination of aggregate elemental composition and quantification by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) is described in Supplementary Table S1. Pulsed medium (S-MEM) or pulsed viral suspension (S-MEM + rAd) were obtained after low voltage pulse delivery (10 x LV) either to the medium or to the viral suspension, in absence of cells. For dose-response study, different fractions of pulsed viral suspension (from 0% up to 100%) were mixed with complementary fractions of unpulsed viral suspension. The protocol used for the analysis of GFP L929 cell conversion as a function of chemical aluminum hydroxide quantities added to the viral suspension is described in Supplementary Figure S4.

Measurement of cell viability.

The percentage of GFP + cells presented in the figures corresponds to the percentage of living cells expressing GFP. The number of living cells for each experimental value was determined by flow cytometry, using the BD Accuri CFlow Plus software. More precisely, cells grown as adherent cultures were washed with PBS to remove all the floating dead cells. Living cells were then detached with TrypLE Express and immediately analyzed by the flow cytometry. Classical forward and side scatter gating was used to identify live cells, while removing debris, cell fragments, pyknotic cells, etc. (see the gating strategy in Supplementary Fig. S2). The Accuri C6 allows the precise determination of the sample volume (using a microprocessor-controlled peristaltic pump) in addition to the fluorescence intensity and cell number for any gated population, allowing thus the absolute cell counts of the live cells. Viability of L929 or CHO-hCAR cells at different time intervals after treatment was expressed as a percentage versus the number of living cells counted in the untreated samples (set at 100% viability).

Long term viability was also appreciated using a colony formation assay. Briefly, after treatment, cells (5x10 6 cells/ml in 50 µl), left 40 min in the cuvette at ambient temperature, were serially diluted in complete medium to obtain about 200 cell colonies per 4 cm-well for the non-treated control, and duplicate wells were filled with 3 ml of the ultimate cell dilution.

After a 6-day incubation at 37 °C in a CO 2 -cell incubator, culture medium was aspirated and wells were gently washed with 2 ml of PBS. Cell colonies were fixed and stained for 10 min with Crystal Violet solution (0.5 g in 80 ml distilled water/20 ml methanol), then washed with distilled water. After complete drying, colonies were counted in each well in duplicate.

Statistical analysis.

Data representative of several independent experiments (n=3 to 6) are presented as means ± SD, with experimental points in duplicate for each independent experiment (except where indicated). For experiments including comparison between various conditions, one-or twoway ANOVA, (or a two-way ANOVA for repeated measures for Figure 1) was applied. A Bonferroni's multiple comparison test or, for experiments comparing all columns versus one control column, a Dunnett's test was used as a post-hoc test. When data failed the variance homogeneity test, a non-parametric analysis was done instead, one-or two-way ANOVA on ranks with subsequent Tukey post-hoc test. *p<0.05 was considered statistically significant.

For experiments with measure of fluorescence intensity, data were previously log 10converted before application of a comparison test. All graphs and statistical analyses were realized with GraphPad Prism 5 and SigmaStat 4.0 softwares, respectively.

Results

Effect of different types of electric pulses on adenovirus-mediated gene transfer.

Adenovirus-mediated gene transfer (AMGT) was assessed by infection of L929 with a rAd encoding the GFP gene. Acquisition of GFP expression by target cells was measured by flow cytometry at 48 h post-infection (Table 1) or by continuous real-time imaging during 35 h (Figure 1). The percentage of GFP + cells and the median of fluorescence intensity (only for flow cytometry analysis) were used as indices of AMGT, itself proportional to the cell penetration of viral particles.

Table 1

First, the effects of several types of electric pulses on AMGT were tested for two cell lines: L929, weakly transduced by Ad5, and CHO-hCAR, which exhibits, on the contrary, an excellent rate of adenovirus infection. In the absence of electric pulses (Table 1, I/UP condition), the GFP conversion rate was only around 47% and the fluorescence intensity 3.30x10 4 RFU, using a MOI of 10 4 vp/cell for L929 cells, in contrast with about 83% and 2.34x10 5 RFU, respectively, using only 50 vp/cell for CHO-hCAR cells. This reflects the low rate of viral penetration inside L929 cells, indicating a low expression of specific viral receptors. In subsequent experiments on L929 cells, the viral MOI was set at 0.5-1x10 4 vp/cell as the optimal magnitude to allow the detection of potential positive or negative effects of electric pulses on rAd cell penetration. The effects of several types of electric pulses on the AMGT and on the cell survival were tested for infected L929 (Table 1a) or CHO-hCAR (Table 1b) cells. Regarding L929, low voltage (LV) pulses alone had a significant increasing effect, both in terms of GFP conversion and cell fluorescence intensity. A minor influence of HV pulses delivered prior to LV ones was mainly observed for the fluorescence intensity, since 98% of cells were already GFP + with LV pulses alone (Table 1a). With the electric parameters used, short term survival was much more affected by LV treatment (around 50% survivors) and even more for HV+LV treatment (8%), than by HV treatment alone (90%). Rather, in CHO-hCAR cells, although cell survival was affected by electric treatments in similar proportions than in L929 cells, the AMGT was not modified by electric pulse application (Table 1b). This is likely because they already exhibit an excellent rate of adenovirus infection at baseline, associated to their high levels of hCAR transgenic expression.

Figure 1

When using a real-time monitoring of GFP cell conversion for 35 h following electric pulses, we confirmed that LV pulses significantly enhanced rAd penetration, while HV pulses alone had no effect unless they were combined with LV pulses (Figure 1a). It should be noted that the detection threshold of the fluorescence emitted by the GFP was much higher (roughly 50-60 times more) for the real-time imaging device camera (see supplementary Fig. S3) than for the flow cytometer detector, explaining the quantitative differences between the results of these two types of experiments. Moreover, the real-time monitoring of the average cell eccentricity showed that LV, and even more HV+LV treatments, in line with cell survival results obtained with the flow cytometry experiments (see Table 1), also transiently impaired the capacity of infected cells to attach to dishes, compared to HV pulse treatment or unpulsed cells (Figure 1b). Indeed, the curves tend to reach a maximal eccentricity value between 0.72-0.82, when untreated cells (UI/UP) achieved full adherence. The delay required to cross the eccentricity threshold of 0.75 was about 17 h for unpulsed controls (+/infection) and the HV condition, about 22 h for the LV condition, and 26-29 h for HV+LV- treated cells (+/-infection). In addition, we observed a delayed onset of measurable GFP conversions for I/HV+LV versus I/LV conditions (see Figure 1a). Therefore, the curve divergence observed 20 h after treatment, could be explained by a disturbed and delayed cell proliferation for I/HV+LV-compared to I/LV-treated cells, resulting in lower dilutions of GFP fluorescence in daughter cells and a higher percentage of GFP + detected at delayed time of experiment. This effect also explains the higher fluorescence intensity per cell observed for I/HV+LV-compared to LV-treated cells, 48 h after electric pulse application in both cell lines (see column I/HV+LV in Table 1). Altogether, these data support the idea that HV pulses are not efficient to enhance AMGT in L929 cells and that LV pulses are required to obtain a significant increase in GFP cell conversion.

Figure 2

Optimization of LV pulses and viral MOI for AMGT enhancement in L929 cells.

GFP conversion of L929 cells was investigated as a function of LV pulse number and intensity. For a given MOI, the rate of conversion was enhanced in the same proportion as the number of pulses and their intensity in a range of 4 kVm -1 up to 10 kVm -1 (Figure 2a and 2b, respectively). However, the increase of GFP cell conversion was achieved at the cost of a decline in cell survival which was, for example, below 50% for 10 pulses at 12 kVm -1 (Figure 2a). Cell survival was also affected by the raise of pulse amplitude, at least above a threshold of about 4 kVm -1 (Figure 2b). Since the rate of GFP conversion did not further increase beyond 10 kVm -1 , we set up this amplitude value for all subsequent experiments.

GFP conversion and fluorescence intensity were then investigated as a function of the viral MOI (Figure 2). The GFP conversion rate was increasing in the same proportion as the viral MOI up to a threshold of 60-70%, for both UP and LV pulse conditions (Figure 2c). 2d) rather showed segmental positive linear regression for UP and LV pulse conditions, with a breakpoint at a MOI value of 2x10 4 and 0.5x10 4 , respectively, otherwise corresponding to 60-70% of GFP cell conversion in both conditions (see Fig. 2c). At the ultimate MOI used, fluorescence intensity was 0.13x10 6 RFU for the UP cells and about tenfold more for LV-pulsed cells (Figure 2d). Thus, when nearly all the cells have been GFP-converted, the LV pulse treatment also increased fluorescence intensity several fold.

Investigations on plasma membrane modifications related to LV pulses.

Initially, we assumed a role for plasma membrane electropermeabilization to explain this result. To investigate this mechanism, we have used the non-permeant dye YO-PRO-1 during cell electroporation using either HV or LV pulses. As shown by flow cytometry analysis in Figure 3a, cell penetration of YO-PRO-1 was induced by HV but not by LV pulses used in our experiments, evidencing that an electroporation process was not involved in the enhanced penetration of rAd in L929 cells. To consider the potential role of an electrophoretic effect exercised particularly by long-lasting pulses, we made a comparison of the effects of LV pulses delivered to L929 cells, either prior or after exposure to viral particles. We found that AMGT was enhanced only when both cells and rAd were subjected to LV pulses (Figure 3b). This result suggested that an electrophoretic effect of LV pulses on viral particles could contribute to the observed effect, but also indicated that direct modifications of the plasma membrane by electric pulses were not critical for the AMGT increase.

Figure 3

Observation, characterization, and quantification of aggregated particulate material.

Since there was no evidence that the enhancement of AMGT by LV pulses was mainly related to plasma membrane modifications, we explored the influence of LV pulses on the culture medium itself. A first step was made by simple observation under a phase-contrast microscope of an aliquot of culture medium subjected to LV pulses or not. We detected a particulate material which formed precipitable aggregates specifically in the pulsed medium (Figure 4a, pictures 1-2). To confirm that the presence of these aggregates was due to the application of electric pulses and was independent of the solution used, we reproduced the same experiment with two other solutions, simpler than the initial culture medium (Figure 4a, pictures 3-4). In each case, we observed the formation of qualitatively similar aggregates after application of LV pulses.

Next, in order to characterize the nature of these aggregates indisputably, the material released in each of the three solutions (S-MEM, PBS and Hepes/NaCl), pulsed or not pulsed (as negative controls), was subjected to a highly sensitive analysis: the Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES). This technique allows both the elemental composition and quantification of the samples (see Supplementary Table S1).

This experiment has established that the aggregates were made of aluminum (hydrogen and oxygen cannot be detected by this method). Aluminum (Al) was found in the different solutions only after application of pulses to the different solutions and the quantity produced were independent of their composition but depended on the number of LV pulses, thus, this metal emanates perforce from the energized electrodes (according to the Faraday's second Law). At pH 7, Al is mainly found as aluminum hydroxide (Al(OH) 3 ), insoluble in water, and aqueous Al 3+ ions, resulting from anodic oxidation during application of pulse, are quickly transformed in Al(OH) 3 when the pH > 4. To confirm this fact in our experimental conditions, we added a control in the ICP-AES experiment where the pellet, obtained after pulse application to the PBS solution and centrifugation, was washed with distilled water to eliminate the dissolved salts (including potential soluble Al 3+ ions). This control showed the same Al content formed (around 0.32 µg for one LV pulse) than the unwashed samples, indicating that Al(OH) 3 is the unique source of aluminum in neutral physiologic solutions (see Supplementary Table S1).

Effect of the aluminum hydroxide aggregates on AMGT increase.

To establish that these metallic particles were involved in the AMGT enhancement, L929 cells were infected using four experimental conditions: 1) viral suspension unpulsed, 2) made with medium pulsed only (formation of aggregates before mixing with rAd), 3) pulsed (formation of aggregates in the presence of rAd), 4) viral suspension mixed with cells and pulsed together (formation of aggregates in the presence of rAd and cells). Moreover, in the three first conditions, viral suspension was finally added to target cells either unfractionated or fractionated by low-speed centrifugation in upper and lower fractions, the latter being enriched in aluminum aggregates of various sizes (Figure 4b). Strikingly, delivery of LV pulses to the viral suspension, or even to the plain medium before virus addition to cells, was sufficient to enhance AMGT compared with unpulsed condition (Fig. 4b, unfractionated inocula). Moreover, in each condition, the benefit was greater for the lower fraction than for the upper one, indicating a specific contribution of aluminum aggregates to viral particle entry (Fig. 4b, fractionated inocula). This interpretation was confirmed by the data obtained when medium and virus were pulsed together: not only with the unfractionated viral suspension AMGT was greater than with previous conditions, but after fractionation, the relative contribution of the lower fraction was also strongly increased, suggesting a role for co-aggregation of virus and aluminum particles resulting from concomitant exposure to electric pulses (see Figure 4c, ratio of the upper fraction to the lower one). An ultimate and large increase was observed when LV pulses were also applied on cells mixed with the viral suspension (Fig. 4b).

Figure 4

Additional steps were taken to confirm the role of Al(OH) 3 particles in the AMGT enhancement. First, we have made an experiment comparing the effects of unipolar LV pulses to those of bipolar LV or unipolar LV pulses with alternated polarity (Table 2). The enhancement of viral penetration was greatly reduced with these two latter pulse types, known to produce less metallic particles than unipolar LV pulses of the same intensity and duration [START_REF] Kotnik | Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses. Part II. Reduced electrolytic contamination[END_REF]. Furthermore, to test a dose-dependent effect of Al(OH) 3 aggregates, we realized infections made with mixtures of pulsed and unpulsed viral suspensions. For each virus MOI, the rate of GFP conversion was proportional to the pulsed fraction size (from 0% to 100%). Each curve reached a plateau whose value was dependent on the MOI, i. e. on the quantity of available viral particles (Figure 5a). However, additional increase in fluorescence intensity was detected even beyond the point where the rate of GFP conversion reached a plateau of almost 100%, for the highest viral MOI used (Figure 5b). This indicated that Al(OH) 3 particles acted both on GFP conversion rate and on cell fluorescence intensity.

Table 2

Figure 5 To verify if aluminum hydroxide from another source (than that from electrolysis reactions) could also have a detectable effect, we realized an additional experiment where we mixed the rAd with Al(OH) 3 from a chemical supplier and analyzed the effect on GFP conversion of L929 cells (see Supplementary Figure S4). We used increasing amounts of Al(OH) 3 , with a range (0.08 to 39 µg) including the quantity observed in the culture medium after one LV pulse train (10 x 20 ms): around 9 µg (see Table S1). Consistently, though lower, we observed an enhanced adenovirus entry in L929 cells with similar quantities of aluminum hydroxide from both origin (compare Fig. 4b with Fig. S4) and the dose-effect ranges were also comparable (compare Fig. 5a with Fig. S4). Finally, the experiment depicted in Figure 6 Experimental conditions

Cell line

Measured variable showed that long-term viability of cells treated with various fraction of pulsed medium was much better preserved than that of cells directly exposed to LV pulses.

Figure 6 Discussion

We investigated whether electric pulses could facilitate cell penetration of large biological objects, as viruses, and chose to evaluate the electrotransfer potential of a rAd encoding GFP by measuring the AMGT efficacy. First, we have indeed observed a substantial increase in AMGT resulting from the application of LV millisecond-(but not HV microsecond-) electric pulses, unlike standard protocols used for DNA electrotransfer.

Second, we tried to understand the underlying mechanisms of this increase. Initially, our hypothesis was that the penetration of viral particles was facilitated by a combination of electropermeabilization and electrophoresis. But the AMGT increase occurred in the LVtreated L929 cells in the absence of their electropermeabilization as demonstrated by the absence of YO-PRO-1 uptake. In contrast, above given thresholds, the LV-increased GFP expression was proportional to the number of pulses applied (total duration of the electric field application) and to their voltage. These data suggested a role for an electrophoresisdependent uptake of viral particles by attracting them towards the charged cells, surprising knowing that the charge carried by the adenovirus capsid is very low. However, an electrophoretic effect hypothesis was also supported by the fact that pulse delivery must be done in presence of the virus to be efficient. It became obvious that the mechanism(s) of AMGT increase were not directly due to an effect of electric pulse on the cells, since LV pulse application on L929 cells, several minutes before rAd infection, could not show any impact on virus entry such as an electro-endocytosis process.

To go further, we analyzed whether the LV pulses could have an effect on the virus, namely on the viral suspension. Strikingly, AMGT was enhanced when viral particles were subjected to electric pulses before they come into contact with the cells, indicating that the main effect of electric pulses was rather a modification of the penetrating agent. Since delivery of the pulses on culture medium, prior to addition of the viral particles, was sufficient to get the AMGT enhancement, we suspected the contribution of a facilitating material released from the electrodes, energized during electric pulse delivery. This was confirmed by the detection, characterization, and quantification of Al in aggregates, reproducibly found in several solutions subjected to millisecond pulses.

Several reports have studied the emissions of metallic ions, mainly as undesirable modifications of the experimental conditions and detrimental effects on target cells [START_REF] Kotnik | Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses. Part II. Reduced electrolytic contamination[END_REF][START_REF] Loomis-Husselbee | Electroporation can cause artefacts due to solubilization of cations from the electrode plates[END_REF][START_REF] Stapulionis | Electric pulse-induced precipitation of biological macromolecules in electroporation[END_REF][START_REF] Tomov | Are the stainless steel electrodes inert?[END_REF][START_REF] Saulis | Increase of the roughness of the stainless-steel anode surface due to the exposure to high-voltage electric pulses as revealed by atomic force microscopy[END_REF][START_REF] Rodaitė-Riševičienė | Release of iron ions from the stainless steel anode occurring during high-voltage pulses and its consequences for cell electroporation technology[END_REF].

In particular, one group has observed that electric pulses induced significant precipitation of biological macromolecules (DNA, RNA and proteins) in electroporation using millisecond pulses [START_REF] Stapulionis | Electric pulse-induced precipitation of biological macromolecules in electroporation[END_REF]. The authors have shown that aluminum electrodes caused greater precipitation than stainless steel electrodes. The suggested mechanism was that positively charged metal ions, immediately during their generation with the pulses, interact with the negatively charged residues of nucleic acids or proteins, neutralizing the repulsive forces between macromolecules and facilitating the aggregate formation. It should be noted, that this mechanism is commonly used at an industrial scale for the wastewater treatment under the term of electrocoagulation. This might lead to possible artifacts and false interpretations of gene electrotransfer efficiency. In our experiments, the formation of large aggregates of aluminum hydroxide (visible under optical microscopy as observed in Figure 4a) is probably due to 1) the use of electric millisecond pulses that induced the production of Al 3+ ions in higher amounts, 2) the neutral pH of the solution used, promoting their rapid and total transformation in precipitable Al(OH) 3 , 3) their concentration in the vicinity of electrodes and the electrophoretic force exercised by repeated millisecond pulses, favoring their aggregation. Overall, the release of aluminum ions in the medium modifying the experimental conditions became an attractive hypothesis. Next, we have shown qualitatively that the AMGT increase detected was mainly due to the presence of the aggregating Al(OH) 3 particles formed during millisecond pulses in the conducting medium (see Fig. 4b-c), and quantitatively, since the effect was directly proportional to the particulate material quantity added to cells (see the dose-effect curve in Fig. 5).

Friedrich and collaborators have concluded that aluminum cuvettes could be used for experiments, provided that the pulse duration time was in the range of 10-100 µs and amplitude below 400 kVm -1 (Al 3+ concentration not exceeding 60 µM in that case). Under these conditions, plasmid electrotransfer in several cell lines (including L929) seemed not disturbed by aluminum particles in the medium [START_REF] Friedrich | High efficiency electrotransfection with aluminum electrodes using microsecond controlled pulses[END_REF]. In our conditions, long term survival of uninfected L929 cells directly treated with LV pulses can be under 60% of non-treated cells (Figure 6). In contrast, we observed a long-term survival above 90% when these cells were exposed to a full dose of Al(OH) 3 particles, formed during LV pulse application to the crude medium (Fig. 6). Thus, the toxicity seems given mainly by direct exposition of cells to LV pulses rather than through an external effect of Al(OH) 3 particles or of other electrolytic species generated by these pulses. The pH modification due to an exposure to electric pulses has been studied: this phenomenon is more particularly important with aluminum electrodes and use of millisecond pulses [START_REF] Saulis | Changes of the solution pH due to exposure by high-voltage electric pulses[END_REF]. Furthermore, pH variations in the electrolytic chamber can exceed 1 to 2 pH units in average and pH fronts are the principal cause of tissue damage near the electrodes [START_REF] Saulis | Changes of the solution pH due to exposure by high-voltage electric pulses[END_REF][START_REF] Olaiz | Tissue damage modeling in gene electrotransfer: the role of pH[END_REF]. The unbuffered change of pH, improved by each pulse, might be one of the factors triggering the cell death. This cumulative harmful impact on survival rate is clearly shown in Figure 2a. In the case of HV+LV pulses, the penetration of electrolytic products inside cells, permeabilized by the HV pulses, might also explain the very low survival rate observed (see Table 1). Several groups have proposed means to reduce these electrolytic contaminations, among others by replacing unipolar rectangular pulses by bipolar pulses [START_REF] Loomis-Husselbee | Electroporation can cause artefacts due to solubilization of cations from the electrode plates[END_REF][START_REF] Friedrich | High efficiency electrotransfection with aluminum electrodes using microsecond controlled pulses[END_REF][START_REF] Saulis | Changes of the solution pH due to exposure by high-voltage electric pulses[END_REF][START_REF] Soo-Yeon | Aminoclay as a highly effective cationic vehicle for enhancing adenovirus-mediated gene transfer through nanobiohybrid complex formation[END_REF]. So, as a complementary approach, we compared the effects obtained when using bipolar or alternate pulses instead of monopolar pulses (Table 2). Since Al 3+ cations are produced around electrodes, we have also speculated that using alternate pulses at 10 Hz frequency would have a similar effect than real bipolar pulses.

Ultimately, we have some clues to understand the intimate mechanism(s) of AMGT improvement. A lower AMGT increase was observed with Al(OH) 3 from chemical origin (compare Fig. 4b andS4), indicating that qualitative factors matter (different sizes of particles or aggregation capacity?). The GFP conversion rate increased proportionately with the quantity of Al(OH) 3 particles in the medium and reached a plateau, proportional to the virus concentration (Fig. 5a). This would mean that (large) aggregates of Al(OH) 3 particles, combined with virions, probably promote more numerous cell encounters (considering the effect of aggregate size, density or/and global charge) than -free‖ virus, resulting in more cells expressing at least one copy of GFP gene. Besides, we have shown that more virions were associated to Al(OH) 3 aggregates when these latter were generated from the viral suspension (Fig. 4b-c). This was probably due to Al 3+ ions reactivity with neighboring viral particles during their generation with the pulses (interaction with the negatively charged residues of viral capsid proteins), associated to their electrophoretic drift towards the virions, leading to precipitable Al(OH) 3 -virion aggregates. Then, the cell fluorescence intensity actually increased with increasing amounts of Al(OH) 3 aggregates and virions, only when nearly all cells had been GFP-converted, i.e. when they had at least one viral particle expressing GFP (Fig. 5b). This could mean that more virions, associated to Al(OH) 3 aggregates, should penetrate together (and/or enter more frequently) per cell with LV pulse delivery, explaining the differences observed between curves, at the highest viral MOI used (Fig. 2d). All together, these results imply that the mechanism of cell penetration is more efficient for the Al(OH) 3 aggregate-associated virus than for the free virus in L929 cells.

Finally, if we compare the latest bar of Fig. 4b with those of the same figure, and the data of

Conclusion

In the cell line and electric conditions tested here, we came to the conclusion that the main contribution of the electric pulses to enhanced adenovirus penetration was not a modification of the target cell but rather a modification of the penetrating agent. We have demonstrated that the increased entry of adenovirus particles in L929 cells after application of LV pulses, essentially resulted from virus association with large aluminum aggregates formed during the electric impulses from aluminum plate electrodes. When virus particles, and even more when both cells and virions are present during the pulses, the electrophoretic effect could also play an important role. It is likely, even if not proven here, that the virus/metallic aggregate complex enters cells through a (or several) endocytosis process(es), including macropinocytosis, considering the large sizes of some aggregates observed. These results should hence be generalized to several types of human cells that are naturally weakly or not transduced by adenovirus vectors, including primary cancer cells, differentiated endothelial or epithelial cells, and mesenchymal or hematopoietic stem cells. Adenovirus 5 receptor (CAR) is frequently deficient in these cells, resulting in low penetration of viral particles. Many attempts to improve adenovirus entry in vitro and in vivo focused on virus complexation with polycations (cationic lipids or polymers), facilitating its binding to negatively-charged cell membrane. But these substances have frequently shown cytotoxic effects. Recently a functionalized magnesium phyllosilicate (aminoclay) as has been shown to enhance AMGT through nanobiohybrid complex formation with Ad particles, aggregating them and making their surface charge highly positive, thereby providing an additional cellular entry mechanism by caveolae-dependent endocytosis [START_REF] Soo-Yeon | Aminoclay as a highly effective cationic vehicle for enhancing adenovirus-mediated gene transfer through nanobiohybrid complex formation[END_REF]. Besides, increasing evidence indicates that some viruses naturally spread also in virion groups such as aggregates or in secreted lipid vesicles. These structures increase the MOI independently to the viral population density and contribute to the maintenance of viral genetic diversity. In their natural hosts, this could have implications for, inter alia, virus transmission, genetic complementation between mutants and innate immunity evasion [START_REF] Sanjuán | Collective infectious units in viruses[END_REF].

What can we learn about plasmid DNA (pDNA) electrotransfer mechanisms from this study? The diameter of the linear form of pDNA is in order of 2-3 nm, while that of the supercoiled pDNA of 4.7 kbp is one order of magnitude larger [START_REF] Escoffre | New insights in the gene electrotransfer process: evidence for the involvement of the plasmid DNA topology[END_REF]. These diameters, unlike that of Ad5, remain largely compatible with a pDNA translocation into cells following an -electropermeabilization‖ process. Ultimately, comparative investigations of electrotransfer for various nano-objects (including other viruses) must be continued for a deeper understanding of internalization processes and safety of future therapeutic use.

Correspondence and requests for materials should be addressed to T.R. *Email: thierry.ragot@gustaveroussy.fr Mailing address: Gustave Roussy, PR2, UMR 9018, 114 rue Edouard Vaillant, 94805

Villejuif, France.

Legend to tables and figures

  Then, the curves flattened to progressively reach a plateau where up to 98% of L929 cells expressed GFP. However, comparing these two dose-response curves from the calculation of their half maximal effective virus concentrations (EC 50 ): EC 50 LV = 0.35x10 4 vp/cell and EC 50 UP = 1.41x10 4 vp/cell, the application of LV pulses resulted in about a four-fold viral MOI reduction corresponding to a boost of the cell transduction efficacy. The fluorescence intensity in relation to the viral MOI (Figure

Fig. 2c /

 2c Fig.2c/2d with those of Fig.5a/5b, respectively, we observe that AMGT was always improved when cells were present during pulse application. This difference can be interpreted by the role played again by the electrophoretic effect of LV pulses, pushing metallic/virus aggregates towards cell surface.

Table 1 .

 1 Comparison of GFP gene expression and survival of L929 and CHO-hCAR cells following adenovirus infection and delivery of various types of electric pulses. a) L929 and b) CHO-hCAR were treated with the same procedures, except for the viral MOI (10 4 vp/cell and 50 vp/cell, respectively). GFP + cells (in % of living cells), median of fluorescence intensity (in log 10 RFU) and cell survival (in % of UI/UP cells) were scored by flow cytometry, 48 h after the treatment. Abbreviations: UI: uninfected, I: infected, UP: unpulsed, LV/HV/HV+LV: application of low, high, high plus low voltage pulses, respectively. Data are presented as means ± SD (with n=6 for L929 and n=3 for CHO-hCAR cells). Statistical significances: *p<0.05, **p<0.01, and ***p<0.001 stand for comparisons of GFP + cells and fluorescence intensity versus I/UP condition; # p<0.05 and $ p<0.05 for the same comparisons versus I/HV or I/LV condition (one-way ANOVA on ranks with Tukey post-test or one-way ANOVA with Bonferroni's post-test for L929 and CHO-hCAR, respectively); °°°p<0.001 for comparison of cell survival versus UI/UP condition (one-way ANOVA with Dunnett's post-test).

  Statistical significances: *p<0.05 and ***p<0.001 stand for comparisons of GFP + cells and fluorescence intensity versus I/UP condition; ### p<0.001 for the same comparisons versus I/unipolar LV condition (one-way ANOVA with Bonferroni's post-test).

Figure 1 .

 1 Figure 1. Real-time monitoring of GFP conversion of L929 cells after adenovirus infection and delivery of various types of electric pulses. a) Percentage of GFP-converted cells as a function of elapsed time (in h), for each type of electric pulse. GFP + cells percentage resulted from the ratio of fluorescent adherent cell number to total adherent cell number assessed by image analysis using the IncuCyte Zoom software. For optimal presentation of the initial stage of GFP expression, Y-axis is broken to indicate the change of scale above 3%. b) Average cell eccentricity variations (from 0 up to 1) as a function of elapsed time (in h), calculated using the IncuCyte Zoom software. These variations reflect adherent cell recovery several hours after treatment. Plateau values of eccentricity (at 0.72, 0.75 and 0.82) are indicated by horizontal dotted lines. Treatment procedures for L929 cells were as indicated in the -Materials and Methods‖ section, with the MOI set at 4x10 4 vp/cell. Image acquisition started 2 h after the treatment (vertical dotted line on the graph a) and was repeated every 3 h for 35 h. Abbreviations and curve symbols: UI: uninfected, I: infected, UP: unpulsed, LV/HV/HV+LV: application of low, high, high plus low voltage pulses, respectively; UI/UP (▬▬), UI/HV+LV (▬•▬), I/UP (•••▲•••), I/HV (---▼---), I/LV (▬■▬), I/HV+LV (-•--•-). Data are presented as means ± SD (with n=3 for each condition). Error bars are indicated either up or down, depending on experimental points, for optimal visualization of the curves. Statistical significances: *p<0.05, **p<0.01 and ***p<0.001 stand for comparisons of GFP + cells in a) and average cell eccentricity in b), at each time interval and between different experimental conditions indicated below both graphs (two-way ANOVA for repeated measures with Bonferroni's post-test).

Figure 2 .

 2 Figure 2. Influence of various experimental parameters on GFP gene expression and survival of L929 cells subjected to adenovirus infection and LV pulse application. GFP + cells (in %) and cell survival (in % of UI/UP cells) as a function of: a) the number (in N) of LV pulses delivered, b) the amplitude of LV pulses (in kVm -1 ). Infected L929 cells (MOI = 10 4 vp/cell) were subjected to (a) increasing numbers of LV pulses at 12 kVm -1 , or (b) 10 LV pulses of increasing voltages up to 12 kVm -1 , 48 h prior to flow cytometry. Abbreviations and curve symbols for a-b graphs: GFP + cell % (▬•▬) with error bars, cell survival (---■---) with error (thin dotted lines). Data are presented as means ± SD (with n(a)=5 and n(b)=3). c) GFP + cells (in %) and d) median of fluorescence intensity (in log 10 RFU), as a function of the viral MOI used for infection (half dilutions from 4x10 4 to 0.25x10 4 vp/cell were converted into a log 2 for graphic considerations). L929 cells were mixed with rAd or with the vehicle alone (UI condition) and left unpulsed (UP condition) or subjected to LV pulses and analyzed by flow cytometry, 27 h post treatment. In c, half maximal effective virus

Figure 3 .

 3 Figure 3. Contribution of electropermeabilization and electrophoretic effect in the GFP conversion enhancement induced by LV pulse application in infected L929 cells. a) Flow cytometry detection of intracellular YO-PRO-1 to assess electropermeabilization of L929 cells subjected to HV versus LV pulse application. L929 cells were exposed to YO-PRO-1, immediately subjected to HV or LV pulses (amplitude = 12 kVm -1 ) and, 40 min later, to flow cytometry analysis (excitation: 488 nm, emission: 530/30 nm). The graph represents cell distribution (count) according to fluorescence (in RFU) by the YO-PRO-1 dye (logarithmic scale). Cells not exposed to YO-PRO-1 (black line, auto-fluorescence), cells exposed to YO-PRO-1 without application of electric pulses (blue line) or subjected to LV or HV pulses (red and green line, respectively). b) GFP + cells (in %) as a function of the order and time interval between LV pulse application and rAd infection. L929 cells were infected with rAd at MOI = 10 4 vp/cell, 5 min before (blue columns) or 1 min (red columns) to 5 min (green columns) after LV pulse delivery. The GFP + cells were scored by flow cytometry 48 h after the treatment. Abbreviations: UI: uninfected, I: infected, UP: unpulsed, LV: application of low voltage pulses. Data are presented as bar chart, with means ± SD (n=5). Statistical significance for b: **p<0.01 stands for comparisons between experimental conditions as indicated by the brackets on the graph (one-way ANOVA on ranks with Tukey post-test).

Figure 4 .

 4 Figure 4. Observation of aggregated particles after LV pulse application and their role in the enhancement of GFP conversion in infected L929 cells. a) Aggregated particle observation by optical microscopy (magnification: × 200), after application of twenty LV pulses on aliquots of three different solutions: 1) negative control (S-MEM unpulsed), 2) S-MEM pulsed, 3) PBS pulsed, 4) Hepes/NaCl pulsed. Size scale corresponding to the black bar is 80 µm. Photo editing was realized with Gimp 2.1 software. b) Role of aggregated particles in the enhancement of GFP gene expression. L929 cells were infected with rAd (final MOI of 10 4 vp/cell) using three types of inoculum: 1) viral suspension made in crude

Figure 5 .

 5 Figure 5. Analysis of GFP gene expression in L929 cells as a function of viral MOI and percentage of the viral suspension treated with LV pulses. S-MEM medium was mixed with rAd to obtain viral suspensions adjusted at three different MOI in vp/cell: 5x10 3 (••••••), 10 4 (---•---), and 2x10 4 (▬▬), when added to L929 cells. Pulsed fractions of the viral suspension (from 2.5 to 100%) were indicated on the X-axis, in log 10 scale for the sake of clarity. They were added to L929 cells that were analyzed by flow cytometry 48 h post treatment. a) GFP + cells (in %), or b) median of fluorescence intensity (in log 10 RFU) are presented. By comparison, the median level of L929 cell fluorescence in the absence of GFP expression (auto-fluorescence of uninfected cells treated with 100% of pulsed medium) was 3.77 ± 2.72 log 10 RFU. Data are presented as means ± SD (n=3). Statistical significances for a and b: *p<0.05, **p<0.01, and ***p<0.001 stand for intra-curve comparisons of GFP + cells and fluorescence intensity versus the -no pulsed fraction‖ condition; ### p<0.001 for comparisons between curves representing different virus MOI (two-way ANOVA with Bonferroni's post-test).

Figure 6 .

 6 Figure 6.Long term viability of non-infected L929 cells treated with increasing fractions of pulsed medium or direct LV pulse application. Culture medium was subjected to LV pulses in the absence of cells. Then, L929 cells (5x10 6 cells/ml) were incubated for 40 min in a mix of crude and pulsed medium with increasing proportions of the latter, through six experimental conditions (0% to 100% PM). They were then diluted in complete DMEM medium and plated into duplicated 4 cm-wells (technical replicates). In one additional experimental condition, cells were directly subjected to LV pulses before the colony formation assay (rightmost column). Colonies were counted in each well after 5 days of culture and counts were expressed in percentages of the control condition (0% PM).Abbreviations: PM: pulsed medium, LV: low voltage. Data are presented as bar chart, with means ± range of technical replicates.

Table 2 .

 2 Comparison of GFP gene expression and survival of L929 cells after adenovirus infection and delivery of various LV pulse types. L929 cells were infected (MOI = 10 4 vp/cell) and subjected to ten LV pulses either unipolar, bipolar, or with alternate polarities, respectively. GFP + cells (in % of living cells), median of fluorescence intensity (in log 10 RFU)

	and cell survival (in % of UI/UP cells) were scored by flow cytometry analysis 24 h ± 30 min
	after the treatment. Abbreviations: UI: uninfected, I: infected, UP: unpulsed, LV: application
	of low voltage pulses, altern.: alternate. Data are presented as means ± SD (n=3).

Acknowledgements

This work has been supported by CNRS, Gustave Roussy and Paris-Saclay. The research was initially conducted in the scope of the EBAM European Associated Laboratory (LEA). It has also benefited from the facilities of the Plateforme d'Imagerie et de Cytométrie (PFIC) -UMS 3655 CNRS/US 23 INSERM, Gustave Roussy Cancer Campus, with technical help of Yann Lécluse. The determination of elemental composition of samples by ICP-AES was realized in the Laboratoire de Planétologie et Géodynamique (LPG) -UMR 6112 CNRS, Nantes University, with the expertise of Carole La and supervision of Antoine Bezos who are warmly thanked. In the lab, we express our appreciation to Lluis Mir for critical remarks on results, Karim Benihoud for helpful discussions, Isabelle Leray, Jean-Rémi Bertrand, and Alain Deroussent for technical discussions, Antoine Azan and Thomas Garcia-Sanchez for their technical help for electrical devices, Pierre Busson and Catherine Brenner for their careful proofreading of the manuscript.

Authors contributions

T.R.: conception and design of the study, performance of biological experiments, preparation of the original draft of the manuscript; F.A. and T.R.: construction and use of electrical devices, data interpretation; A.T. and T.R.: data analysis, statistical study, writing and final approval of the manuscript. All authors reviewed the manuscript.

Additional information

Competing interest: The authors declare they have no competing interest.


domain: chim sdv

text:
Sylvain Guyot

Fractionnement et analyse chromatographique des tanins condensés de la pomme

Sylvain Guyot

•

  Photo-protecteur UV • Anti-appétant vis-à-vis des insectes • Régulation redox, protection contre stress oxydant Propriétés dans les fruits et leurs produits (jus, cidres, vins,…) Et de nombreuses applications alimentaires et non alimentaires (tannage du cuir, agents clarifiants, bases pour substituts de résines époxy, …) en fonction de leur taille ? NB: La chromato d'exclusion stérique « classique » ne fonctionne pas….trop de phénomènes d'adsorption avec les gels classiques La chromatographie en phase normale (Si-OH) …..pour les oligomères jusqu'à DPn 7de CH 2 CL 2 , MeOH, H 2 O La chromatographie en phase inverse (C18) …..pour les polymères jusqu'à…DPn >200 Gradient d'H 2 O et ACN D'après Guyot et al., J. Agric. Food Chem., 2001 DPn =

Procyanidines, tanins et impact gustatif : Structure et concentration pour un équilibre entre amertume et astringence Journée d'animation du Club Chromato de l'Ouest -Nantes -Le 10 mars 2020 Journée d'animation du Club Chromato de l'Ouest -Nantes -Le 10 mars 2020

Spectrométrie de masse des tanins condensés


text:
Sophie Guilois-Dubois

Sylvain Guyot

Pascal Poupard

Preparative isolation of apple Flavan-3-ols by pH-zone-refining centrifugal partition chromatography combined with reversed-phase liquid chromatography

Introduction

Flavan-3-ols (which include procyanidins and condensed tannins) are widespread polyphenols in fruits and edible plants and having numerous properties. They are largely responsible for astringency and bitterness in cider beverages. To study their sensorial, nutritional and biological features, it is essential to recover pure and native flavan-3-ols fractions. A gentle strategy combining pH-zone-refining centrifugal partition chromatography (pH-ZRCPC) and preparative reversed-phase liquid chromatography (Prep-RPLC) was developed to purify hundred milligrams of apple flavan-3-ols fraction in a highly purified state.

Conclusion

Starting from a crude apple polyphenol extract, our experimentations demonstrate the relevance of using CPC combined with a pH-displacement mode to efficiently separate flavan-3-ols from hydroxycinnamic acid derivatives at a preparative scale. The objective was successful through optimisation of solvent systems with suitable conditions for pH-ZRCPC and an attention paid to avoid autoxidation. Finally, preparative reversed-phase chromatography allowed the separation of the flavan-3-ols with satisfactory purity (82%) and recovery (73%). The composition of the collected fraction (called MM-FA) was characterized and compared to that of the crude extract (Fig. 3) showing that it contained only flavan-3-ols (Table2).

Sophie Guilois

Analyses of fractions using UPLC-UV/MS

pH-zone-refining CPC was carried out using a FCPC200® apparatus (Kromaton Technologies) (Fig. 1). The collected CPC fractions corresponding to flavan-3-ols (FA), dihydrochalcones (DHC) and flavonols (FO) were immediately acidified to avoid oxidation, and pooled. Prep-RPLC was performed on this intermediate freeze-dried fraction with a Lichrospher 100 RP-18, 12 µm column (Merck, Darmstadt, Germany) and isocratic mode, to recover only flavan-3-ols (Fig. 2). The pH-ZRCPC fractionation was optimized from a crude cider apple polyphenol extract (named TotPP) obtained from a non-oxidized juice of the French cultivar Marie Menard (Millet et al. [1]). it contained essentially flavanols monomers and oligomers (46.3%) with an average degree of polymerization close to 3.3, as measured by phloroglucinolysis coupled to reversed-phase HPLC), hydroxycinnamic acid derivatives (35.9 %) and dihydrochalcones (2.6%) (Table 2).

pH-ZRCPC + Prep-RPLC procedures

Optimization of two-phase solvent system for pH-ZRCPC

The partition coefficients (K) of the target compounds for a conventional CPC and pH-ZRCPC (by adding of a base or an acid) were determined for four biphasic solvent systems adapted from OKA [2] composed of ethyl acetate:n-butanol:water with 5:0:5, 3:2:5, 2:3:5 and 1:4:5 (v:v) named systems A, B, C and D, respectively. For successful separation using pH-ZRCPC, it is necessary to respect Kbase << 1 and Kacid >> 1, for an acidic analyte. This allowed to select system B as the best one to discard hydroxycinnamic acid derivatives (HCA)(Table 1).

TotPP fraction

MM-FA purified fraction

[ 1 ]

 1 Millet, M, et al., Food Chem., 2019, 276, 797. [2] Oka, F, et al., Journal of Chromatography, 1991, 538, 99.

  -Dubois a,c , Sylvain Guyot a,c , Pascal Poupard b,c a INRAE UR1268 BIA -Polyphenols, Reactivity, Processes, F-35653 Le Rheu, France b IFPC (French Institute for Cider Production), F-35653 Le Rheu, France c UMT ACTIA Nova 2 Cidre, F-35653, Le Rheu, France Table 2. Composition of phenolic compounds in TotPP and MM-FA extracts (results expressed in g/Kg of dry extract) and structural characterization of flavan-3-ols.

Fig. 3 .

 3 Fig.3. Comparison of analytical reversed-phase chromatographic profiles at 280 nm of the TotPP extract (red) and the final purified flavanol extract (black).

Fig 1 .

 1 Fig 1. pH-ZRCCC chromatogram of the crude sample from 1 g TotPP extract. solvent system used: ethyl acetate-n-butanol-water (3:2:5); flow rate: 5 mL/min; revolution: 1200 rpm. Retainer (TFA) and eluter (NaOH) at 10 mM in the stationary and mobile phases, respectively.


text:
Claudia Mariana

Castillo Fraire

Pascal Poupard

Sophie Guilois-Dubois

Erika Salas

Susana Soares

Elsa Brandao

Victor De Freitas

Sylvain Guyot

Claudia Mariana Castillo Fraire

Enzymatic synthesis, structures, interactions with saliva proteins and quantification in juices of a series of dehydrodicaffeoylquinic acids, one of the main classes of oxidation products in apple-based beverages

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Mainly located in the flesh and in the skin, apples polyphenols show a great diversity of molecules belonging to the classes of hydroxycinnamic acids, catechins, procyanidins oligomers and polymers, dihydrochalcones, flavonols and anthocyanins. When apple are processed into juices and ciders, a great diversity of newly formed polyphenolic molecules is generated by enzymatic oxidation. Some of those oxidation products contribute to the colour of the juice. However, a great majority are colourless and their detailed structures, their real concentrations in the juices or their contribution to sensory, antioxidant and other nutritional properties in the final products are still scarcely known. Previously, we showed that newly-formed products resulting from enzymatic oxidation of 5′-O-caffeoylquinic acid (CQA), also commonly known as chlorogenic acid, still presented high antioxidant activity in vitro [1]. However, we have no information regarding their possible involvement in the bitter taste and the sensation of astringency that may be related to the presence of polyphenols in fruit-derived beverages. We remember that, in this particular case, astringency is a consequence of the precipitation of salivary proteins due to the tanning properties of polyphenols. We present here our main recent results related to the oxidation products of CQA focusing on their UV, MS and NMR structural characterisation, their LC-MS quantification in a panel of commercial juices and their capacity to aggregate salivary proteins.

MATERIALS & METHODS

Synthesis, purification and structural analysis: DDCQAs were synthesized from CQA in model solution using a crude apple polyphenoloxidase (PPO) extract. Ten DDCQAs were purified by centrifugal partition chromatography and RP18 HPLC at the semi-preparative scale [2]. Their complete structural elucidation was achieved by MS and 1D and 2D NMR 1 H and 13 C.

Quantification in apple juices: a new LC-UV-MS method was developped using the purified standards to quantify native polyphenols, the series of DDCQAs and a series of CQA-epicatechin dimers in 45 commercial and experimental apple juices.

Aggregation to salivary proteins (SP): a mixture of soluble DDCQAs was incubated with acidic saliva. After centrifugation, the supernatant was analyse by RP18 HPLC-UV and compared to the initial mixture in order to quantify the remaining SP and DDCQAs [3].

RESULTS & DISCUSSION

A series of ten dehydrodicaffeoylquinic acids (DDCQAs) were synthesized by enzymatic oxidation of a model solution of 5′-O-Caffeoylquinic acid (CQA), using a crude apple polyphenoloxidase extract. Then, they were purified by centrifugal partition chromatography and RP-18 HPLC at the semi-preparative scale [2]. Their complete structural elucidation was achieved by 1D and 2D NMR 1 H and 13 C revealing original dihydrobenzofuran, benzodioxan and dihydronaphtalen polyphenolic skeletons. In addition, for the first time a new symmetric structure exhibiting two free catechol groups was identified.

The capacity of DDCQAs to aggregate salivary proteins (SP) was studied using a precipitation method associated to HPLC analysis of the supernatant [3]. Experiments were conducted in model solutions in order to vary at the same time the concentrations and the ratio of both polyphenols and saliva proteins in the medium. Results showed that interactions between salivary proteins (SP) and DDCQAs are highly impacted by the ratio SP/DDCQAs, and in the tested conditions, we hypothesize that a low number of DDCQAs is necessary to significantly precipitate SP. Moreover, a specific interaction of DDCQAs with cystatins and statherin/PB was observed during these experiments. Some of these observations were confirmed by fluorescence quenching analysis. Surprinsingly, DDCQAs exhibited weak interactions with PRPs (Proline Rich Proteins) but interactions were much more significant with statherins/P-B peptide and cystatins.

Finally, polyphenol oxidation products, including the DDCQAs series completed by some epicatechincaffeoylquinic acid dimers, were quantified for the first time in real juices by developing a new LC-MS method. Its application to 32 commercial, 13 experimental and 9 craft apple juices revealed that these families of compounds can reach several dozen mg/L apple juice contributing up to 14 % of total polyphenols.