176 Original scientific paper  MIDEM Society Lithography-Free, Crystal-Based Multiresonant Lamb Waves for Reconfigurable Microparticle Manipulation Amgad R. Rezk and Leslie Y. Yeo Micro/Nanophysics Research Laboratory, School of Engineering, RMIT University, Melbourne, Australia Abstract: Acoustic wave microfluidic devices, in particular those that exploit the use of surface acoustic waves (SAWs), have been demonstrated as a powerful tool for driving microfluidic actuation and bioparticle manipulation. A limitation of these devices, however, is the requirement for the fabrication of interdigital transducer electrodes on the piezoelectric substrate, which upon excitation of an AC electrical signal at resonance, generates the SAW. Not only is the lithographic fabrication a costly and cumbersome step, the necessity for driving the interdigitated transducers (IDTs) at resonance means that the device typically operates at a single frequency at its fundamental resonant state; the higher harmonics that may be available are often weak and negligible. As such, reconfiguring a device for different operating frequencies is usually difficult and almost always avoided. Here, we show a Lamb wave device which can mimic the microfluidic actuation and particle manipulation of SAW devices, but which can be fabricated without requiring any lithographic procedures. Moreover, we show that a large number of resonances are available, whose modes depends on harmonics associated with the substrate thickness, and, in particular, demonstrate this utility briefly for reconfigurable particle patterning. Keywords: acoustics; microfluidics; particle manipulation; concentration Generiranje multi-resonančnih Lamb akustičnih valov za manipulacijo bioloških delcev Izvleček: Mikrofluidne platforme ki uporabljajo za aktuacijo zvočno valovanje ter še posebej tiste, ki izkoriščajo uporabo površinskih zvočnih valov (angl surface acoustic waves - SAW.), so se izkazali kot zelo primerno orodje za manipulacijo bioloških delcev. Omejitev pri teh napravah je zahteva za izdelavo prstastih elektrod, t.j. elektroakustičnih pretvornikov na osnovni piezoelektrični podlagi, ki ob vzbujanje z izmeničnim električnim signalom v resonanci generirajo površinske akustične valovanje. Uporaba fotolitografskega postopka pri izdelavi vzbujevalne prstaste elektrodne strukture je eden bolj zahtevnih korakov. Poleg tega prstasta struktura deluje le na osnovni resonančni frekvenci, višji harmoniki, ki so lahko na voljo, pa so pogosto šibki in zanemarljivi. Preoblikovanje prstastih struktur, ki bi omogočalo delovanje na različnih delovnih frekvencah na isti mikrofluidni platformi je ponavadi težko izvedljivo in se ne uporablja. V tem prispevku bomo predstavili enostaven način generiranja Lamb valovanja, ki omogoča transport in manipulacijo bioloških delcev na piezoelektrični podlagi in ga je možno realizirati brez fotolitografskega postopka. Poleg tega bomo v prispevku pokazali, da je možna vzpostavitev različnih resonančnih frekvenc in višjih harmoničnih komponent v povezavi s spreminjanjem debeline substrata. Obenem bomo demonstrirali uporabnost takega pristopa za poljubno manipulacijo delcev. Ključne besede: akustični valovi; mikrofluidika; manipulacija delcev; koncentracije * Corresponding Author’s e-mail: leslie.yeo@ rmit.edu.au Journal of Microelectronics, Electronic Components and Materials Vol. 46, No. 4(2016), 176 – 182 1 Introduction The ability to manipulate (e.g., concentrate, sort and separate) particulate suspensions, particularly those pertaining to biological substances such as cells, is indispensable to microfluidic operations, where appli- cations pertaining to biomedicine and biotechnology such as point-of-care diagnostics, biosensing, drug 177 development, drug delivery and tissue engineering feature prominently [1-3]. Many microfluidic strategies have been proposed for manipulating such micropar- ticles, including the use of inertia [4], electrokinetic [5], optical [6], magnetic [7] and acoustic [8] forces, among others. While many bulk acoustic microfluidic methods for par- ticle manipulation have been proposed [8], their long wavelengths associated with the kHz order frequen- cies typically employed, and the necessity for large ul- trasonic transducers place considerable limitations to miniaturisation and integration into chip-scale devices. More recently, surface acoustic waves (SAWs), which are the high frequency (MHz order) surface counter- part of bulk ultrasonic waves, have been shown to be an extremely powerful tool for microfluidic actuation and particle manipulation [9-12]. In the latter, standing SAWs have primarily been used for trapping particles at nodal or antinodal positions in microchannels for a myriad of applications including single cell and organ- ism focusing, patterning and separation [13,14], even in three-dimensions [15,16]. Additionally, travelling SAWs have also been recently shown to be highly use- ful for particle manipulation [17-19]. Besides the above, which rely on the acoustic radiation force imparted on particles to manipulate them, particle concentration and sorting have also been demonstrated by exploit- ing the hydrodynamic drag force imparted on the par- ticle by SAW-driven microcentrifugation flows [20-23]. Nevertheless, high frequency (MHz order) SAWs re- quire cumbersome processes associated with lithogra- phy, typically achieved by metal deposition, photore- sist coating, UV-exposure and wet etching, in order to fabricate the interdigitated transducers (IDTs) on the piezoelectric substrate that are required to generate the SAWs. These long multi-step procedures are not only time consuming and require skilled technicians, but also involves the additional expense of operating within a cleanroom environment. Moreover, as the SAW frequency is increased closer towards GHz or- der [24,25], the IDT dimensions become progressively smaller given that they correlate with the SAW wave- length, necessitating even more elaborate fabrication such as electron beam or nanoimprint lithography if robust devices are desired. Furthermore, once a device is fabricated, it typically runs on a single SAW resonant frequency, thus limiting many potential microfluidic applications, especially for reconfigurable particle ma- nipulation within liquid drops. We note the possibility of exciting multiple frequencies on a SAW device us- ing a single tapered IDT (TIDT) design [26] (also known as slanted-finger interdigital transducers (SFITs) [27]), although the limitation with such devices is that the width of the SAW that can be produced for a given fre- quency is confined to a lateral dimension that is on the order of its wavelength—typically too narrow to drive any meaningful microfluidic actuation on the scale of a droplet or channel in most cases. Here, we briefly demonstrate the possibility for on-chip reconfigurable microparticle actuation using a sim- ple fabrication step that does not require lithographic procedures. This extends on our previous work that showed, quite counterintuitively and contrary to long- standing practice, that it is possible to generate Lamb waves for microfluidic actuation on a piezoelectric (lithium niobate; LiNbO3) substrate simply with the use of aluminium foil placed in contact with the substrate [28-30]. 2 Method Figure 1: (a) Schematic illustration and (b) images of various electrode patterns on the lithography-free LiNbO3 chip. (c) Laser Doppler Vibrometer (LDV) scan of the chip at 3.5 MHz; the scale bar denotes a length of approximately 1 mm. The simple lithography-free setup is shown schemati- cally in Figure 1a, wherein either conductive silver paste was spotted, or an arbitrary electrode pattern (such as the straight, L-shaped or circular patterns in Water DropSilver Paste Piezoelectric substrate (a) (b) (c) 0-50 50 (nm) A. R. Rezk et al; Informacije Midem, Vol. 46, No. 4(2016), 176 – 182 178 Figure 1b) comprising a conductive aluminium layer deposited with the aid of masking tape, onto the LiN- bO3 chip. To generate the Lamb wave in the substrate, we simply connect these electrodes to an AC source at an appropriate resonant frequency, a number of which exists, as will be discussed below. To demonstrate the possibility for reconfigurable microparticle manipula- tion, we placed a 5 µl sessile liquid drop comprising 3 µm polystyrene particles suspended in water at a concentration of 105 onto the device and observe its behaviour as a function of the applied frequency using a DSLR camera (Canon D50, Tokyo, Japan) which was connected to a long working distance microscopic lens (K2/SC, Infinity, Boulder, CO). The mechanical vibration displacement was acquired using laser Doppler vibro- metry (LDV; MSA-400 and UHF-120; Polytec, Germany) whereas electrical characteristics of the device, namely the insertion loss parameter S11, was measured using a vector network analyser (VNA; ZNB4, Rohde & Schwarz, Germany). 3 Results & Discussion Figure 2: (a) Results from the VNA frequency sweep to measure the insertion loss parameter S11 of the device, which clearly shows multiple resonances starting from the fundamental resonance at 3.5 MHz, as determined from the magnification of (b) the magnitude and (c) the phase angle, and higher harmonics at every additional 7 MHz. The VNA measurement for the insertion loss parameter S11 in Figure 2 shows that the device possesses multiple resonant frequencies, starting from the fundamental frequency at 3.5 MHz, and additional higher resonant modes at increments of approximately every 7 MHz. While we only show results for the frequency sweep across a range from 1 to 50 MHz in Figure 2 for clar- ity, discernible resonant peaks were observed even be- yond 300 MHz. It is worth noting, that these resonances are insensitive to the electrode shape (dot, straight, L-shaped, circular, etc.) or their dimension, since the electric field, which is coupled to the mechanical field, traverses vertically through the chip thickness; the res- onances are therefore only sensitive to the thickness of the substrate. Reducing this by lapping the device us- ing a rotating wheel covered with abrasive slurry led to increases in the fundamental resonant frequency. In particular, given the speed of sound through LiNbO3 c = 3500 m/s and for a substrate thickness T = 500 µm, the fundamental resonant frequency that arises can be computed from f = λc in which the wavelength λ/2 corresponds to T such that f = 3.5 MHz. Subsequent higher harmonics can then be obtained for 3λ/2, 5λ/2,…,(2n+1)λ/2 (n = 0, 1, 2,…) leading to resonances at every interval of 7 MHz, i.e., 10.5 MHz, 17.5 MHz,…, etc. We also note that the proposed chip configuration in Figure 1 possesses an outstanding quality factor Q of approximately 13,000 (in contrast to that of typi- cal SAW devices where Q factors as low as 100 are not uncommon), which not only makes it a very efficient device for microfluidic manipulation, as will describe later, but also renders it as a very sensitive platform for biosensing. Given the optical smoothness of the LiN- bO3 substrate and the uniformity of its thickness, which sets the wavelength, it is perhaps of little surprise that the resonant band (and hence the high quality fac- tor) is very sharp and almost comparable to those of quartz crystals. Such crystals with high quality factor, although extremely sensitive to mass loading (which makes them very efficient sensors), however possess extremely low electromechanical coupling coefficients kt 2, and therefore, a low figure of merit FOM = Qkt 2. Re- markably, however, the present device, when excited at the fundamental resonant frequency, shows a rea- sonable value for kt 2 of 0.8%. Although other piezoelec- tric substrates such as lead zirconium titanate (PZT) possess kt 2 values up to 20%, we note that the FOM = 13,000 x 0.8% ≈ 10,000 surpasses any other piezoelec- tric material we know of; for example, a typical SAW on 128 YX LiNbO3 with Q ≈ 100 and kt 2 = 5.3%, has an FOM ≈ 500, considerably below that here. This suggests that the Lamb wave device is not just simple and low cost to fabricate, but also satisfies a rare requirement for any   Frequency (Hz) 0 1e+7 2e+7 3e+7 4e+7 5e+7 6e+7 S1 1 (d B) -35 -30 -25 -20 -15 -10 -5 0 5 Frequency (Hz) 3.590e+6 3.591e+6 3.592e+6 3.593e+6 3.594e+6 S1 1_ Lo ss (d B) -35 -30 -25 -20 -15 -10 -5 0 Q = 13.059K Frequency (Hz) 3.590e+6 3.591e+6 3.592e+6 3.593e+6 3.594e+6 S1 1_ Ph as e ( de gr ee ) -200 -150 -100 -50 0 50 100 150 200 K2 = 0.0088 (a) (b) (c) - - - - - - S 1 1 ( dB ) 0 10 20 30 40 50 Frequency (MHz) S 1 1 ( dB ) 0 -10 -20 -30 -3.590 -3.592 -3.594 Frequency (MHz) 200 10 0 -10 -20 S 1 1 ( de gr ee ) -3.590 -3.592 -3.594 Frequency (MHz) (a) (b) (c) A. R. Rezk et al; Informacije Midem, Vol. 46, No. 4(2016), 176 – 182 179 piezoelectric substrate of being both a very sensitive as well as a very effective actuator. Upon excitation of the device at the fundamental reso- nant frequency, i.e., 3.5 MHz, standing Lamb waves in the form of a checkerboard pattern are generated in the device with a wavelength of 1000 µm as seen in Figure 1c. We note that the presence of the liquid drop will distort this standing wave pattern due to its mass loading given that the liquid thus acts as a sink in absorbing the waves along its circular periphery [29]. Unlike previous investigations where we showed that these Lamb waves led to the generation of poloidal flows in the drop, which, in turn gave rise to the forma- tion of toroidal particle rings [29,30], the low excitation powers (<50 mW) primarily used in this study rendered acoustic streaming in the drop insignificant, and, as such, the particles suspended in the liquid were only subjected to acoustic radiation forces instead of the dominant hydrodynamic drag. Figure 3: (a) Top and (b) side view of the three-dimen- sional concentric particle patterns that arise under Lamb wave substrate excitation of a 5 µl sessile liquid drop comprising 3 µm particles suspended in water at the fundamental resonant frequency, i.e., 3.5 MHz. The spacing between the concentric rings is approximately 270 µm. Scale bars denote a length of approximately 1 mm. Figure 4: Top view of the three-dimensional concentric particle patterns that arise under Lamb wave substrate excitation of (a) a circular, and, (b) a roughly square 5 µl sessile liquid drop comprising 3 µm particles suspend- ed in water at 10.5 MHz. The spacing between the con- centric rings is approximately 90 µm. Scale bars denote a length of typically 1 mm. Figure 3 shows the resultant particle patterns that arise on the free surface of the drop as well as within its bulk due to their alignment along nodal lines or planes of the standing acoustic wave that is generated as a con- sequence of the radiation force it imparts on the par- ticles; the standing wave arising due to the reflections at the drop boundaries. The concentric patterns, in particular, reflect the radial nodes associated with the breathing mode of the drop, and the 270 µm spacing between the concentric particle rings correspond to a separation length of λ/2, as expected. What distinguishes the present devices from other acoustic devices, however, is the possibility of trigger- ing a large number of higher harmonic frequencies with reasonable efficiency, thus allowing the possibil- ity of reconfiguring the spacing and assembly of the particle patterns in situ. As a brief example, Figure 4a shows the concentric particle patterns when a higher harmonic (10.5 MHz) is excited in the same device (a) (b) (a) (b) (a) (b) (a) (b) A. R. Rezk et al; Informacije Midem, Vol. 46, No. 4(2016), 176 – 182 180 wherein the spacing between the concentric rings has decreased to approximately 90 µm, again correspond- ing to λ /2 at this harmonic frequency. Higher harmon- ics led to smaller spacing, although the pattern cannot be resolved spatially while imaging the whole drop and is hence not shown. Such reconfigurability is in sharp contrast to SAW devices, whose excitation relies on the IDT dimensions, or bulk acoustic wave (BAW) devices in which the excitation is achieved by coating electrodes on the top and bottom surface of the piezoelectric ce- ramic, e.g., PZT. In those cases, the resonant excitation is predominantly limited to the fundamental resonant frequency set by the IDT (in the case of the SAW) or the substrate thickness (in the case of the bulk piston vibra- tion). Although, theoretically, PZT can support higher harmonics effectively, the poor surface roughness of the material leads to much broader resonance (and hence low Q of approximately 100 and even down to about 10) for the higher harmonics, and therefore the coupling is extremely ineffective. To illustrate that the standing waves within the drop that arise due to the reflection off its boundaries, and that it is these that assume the dominant role in de- termining the particle pattern rather than the under- lying vibration pattern on the substrate, we rendered the LiNbO3 substrate surface hydrophobic (with a Tef- lon coating) save for a roughly square region (with the aid of a mask during coating) on which we placed the drop. As seen in Figure 4b, the resulting particle pattern within the roughly square shaped drop is altered and closely resembles the drop contour. Finally, we demonstrate the possibility of efficiently generating acoustic streaming, and, in particular, mi- crocentrifugation flows, in these devices even at these higher harmonics, which cannot be achieved either us- ing the SAW or BAW devices given the extremely weak higher harmonics in those devices, particularly given the ineffective higher harmonic coupling evident (both Q and kt 2 exponentially decay with frequency). Figure 5 shows the particle concentration as a consequence of the azimuthal acoustic (Eckart) streaming within the drop, whose underlying mechanism we have discussed elsewhere [20-23], when the substrate is excited at much higher harmonics, i.e., 150 MHz, at large applied powers (approximately 500 mW) that give rise to larger substrate vibration displacements. In addition to the large streaming velocities, typically on the order of 0.5– 1 mm/s observed, it can be seen that the bipolar vor- tex gave rise to particle concentration in two distinct islands. The ability to trigger these higher frequencies even with such a simple device then constitutes a con- siderable advantage given the need to date for elabo- rate lithographic procedures (e.g., electron beam or na- noimprint lithography) to fabricate sufficiently robust electrodes for high frequency operation [24,25]. (a) (b) (c) Figure 5: Bipolar vortex acoustic streaming flow is generated in a 5 µl sessile liquid drop comprising 3 µm particles suspended in water by exciting vibration in the substrate at a harmonic resonant frequency of 150 MHz with large input powers (approximately 500 mW). This leads to rapid concentration of the particles into two particle islands through particle shear induced mi- gration [20-23]. The left image (a) shows the drop prior to excitation at time t = 0 wherein the particles are dispersed throughout the drop, the centre image (b) shows the bipolar vortices in the drop at t = 1 s during Lamb wave excitation, and the right image (c) shows the drop immediately after excitation at t = 5 s wherein the particles have concentrated and the substrate vi- bration relaxed. Scale bars denote lengths of approxi- mately 0.5 mm. 4 Conclusion By exploiting the large number of resonances associ- ated with the Lamb wave vibration through the thick- ness of a substrate comprising a piezoelectric material, namely, lithium niobate, and its extremely high quality factor and hence figure of merit, we show the possibil- ity of reconfigurable microfluidic actuation and parti- cle manipulation on a single device. 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Rezk et al; Informacije Midem, Vol. 46, No. 4(2016), 176 – 182 183 Original scientific paper  MIDEM Society Microfluidics-Directed Self-Assembly of DNA- Based Nanoparticles Guillaume Tresset1, Ciprian Iliescu2,3 1Laboratoire de Physique des Solides, CNRS, Univ. Paris-Sud, Université Paris-Saclay, France 2National Institute for Research and Development in Microtechnologies, IMT-Bucharest, Bucharest, Romania 3Academy of Romanian Scientists, Bucharest, Romania Abstract: The ‘bottom-up’ paradigm of nanofabrication mostly relies on molecular self-assembly, a process by which individual components spontaneously form ordered structures with emerging functions. Soft nanoparticles made up of therapeutic DNA condensed by cationic lipids or surfactants hold a great potential for nonviral gene delivery. Their self-assembly is driven by strong electrostatic interactions. As a consequence, nanoparticles formulated in bulk often exhibit broad size distributions not suitable for practical delivery applications. We will review the recent strategies we developed to control the self-assembly kinetics by using microfluidic devices. This combined approach may open attractive opportunities for the directed self-assembly of complex soft nanomaterials in particular for biomedical purposes. Keywords: microfluidics; self-assembly; DNA; nanoparticle; nonviral gene delivery Nadzorovano samourejanje DNA nanodelcev na osnovi mikrofluidike Izvleček: Paradigma nanoizdelave „od spodaj navzgor” (angl. ‘bottom-up’) v glavnem temelji na molekularni samosestavljanju oziroma samourejanju, to je procesu, s katerim posamezne komponente spontano tvorijo urejene strukture s specifičnimi funkcijami. Mehki nanodelci, sestavljeni iz terapevtskih DNK, dobljenih z metodo kondenzacije kationskih lipidov ali površinsko aktivacijskih snovi (surfaktantov), predstavljajo velik potencial za nevirusno dostavo in vnos genov. Njihovo samourejanje je posledica močne elektrostatične interakcije. Posledica tega je, da imajo nanodelci, ki s samourejanjem tvorijo kompleksne strukture, pogosto široko porazdelitev velikosti, kar pa ni vedno primerno za praktične aplikacije. V članku je podan pregled razvoja novih strategij za nadzorovan proces kinetike samourejanja s pomočjo uvedbe mikrofluidnih pristopov, s katerimi lahko odpravimo zgornjo pomanjkljivost. Predstavljeni novi kombinirani pristopi omogočajo kontrolirano samo-sestavljanje kompleksnih mehkih nanomaterialov, zlasti primernih za biomedicinske namene. Ključne besede: mikrofluidika; nanodelci; samourejanje; DNA; nevirusni vnos genov * Corresponding Author’s e-mail: guillaume.tresset@u-psud.fr Journal of Microelectronics, Electronic Components and Materials Vol. 46, No. 4(2016), 183 – 189 1 Introduction Microfluidics is a developing field with applications covering tissue engineering [1-3], cell analysis [4-7], drug discovery [8-9], bioassays [10] and chemical syn- thesis [11-15]. Technology has arrived at a stage where it is now possible to handle and to shape the molecu- lar constituents of matter with nanometer accuracy, whether they are inorganic or biological. As George M. Whitesides put it [16], “the physical sciences offer tools for synthesis and fabrication of devices for measuring the characteristics of cells and sub-cellular compo- nents, and of materials useful in cell and molecular biol- ogy; biology offers a window into the most sophisticat- ed collection of functional nanostructures that exists.” Two paradigms have emerged for the fabrication of nanometer-scaled materials: the ‘top-down’ approach – widely used in the microelectronics industry through lithography – enables to pattern bulk materials such as silicon with features size down to one nanometer and with high batch-to-batch repeatability. The technol- 184 ogy is limited so far to two-dimensional structures at the surface of a substrate. The ‘bottom-up’ approach in turn has been successfully exploited by nature to build up the most complex systems with high throughput, namely, living organisms. The limitation mostly arises from our inability to tune the interactions between constituents in such a way that they self-assemble into desired structures in a repeatable manner. Our present scientific knowledge gives us access to only a small set of architectures and functions, while nature has benefited from billion years of evolution to learn how to make the most elaborate devices such as the human brain with a low error rate. A third paradigm is subsequently emerg- ing and consists of combining the two others. In other words, it aims at fabricating complex three-dimensional structures via self-assembly with high reproducibility. DNA-based nanoparticles are such complex structures and hold a great potential in medicine. Their architec- ture and their function are inspired from virus in the sense that they carry genetic information encoded in compacted nucleic acids – either DNA or RNA – in view of its delivery into target cells [17]. As a matter of fact, a number of viruses have been engineered in such a way that they deliver therapeutic genes with the ef- ficiency of a viral infection. Indeed, the regular func- tion of a virus is to inject its genes into an infected cell, which will then express viral proteins and nucleic ac- ids to make up new viruses. The strength of viruses is that they can circulate inside an organism while being not recognized by the immune system and targeting specific cells. However, they can induce inflammatory responses and provoke cancer through uncontrolled gene insertion. By contrast, nonviral vectors are safer and more versatile than engineered viruses, even though their efficacy is still insufficient. The objective of nonviral gene delivery [18] is therefore to devise na- nometer-scaled synthetic particles containing nucleic acids to deliver into specific cells with high efficacy. The particles must be nontoxic, easy to fabricate, and with excellent batch-to-batch repeatability. This article reviews our recent works on the self-assem- bly of DNA-based nanoparticles for use in nonviral gene delivery. It shows in particular how the third paradigm of nanofabrication can be used through different microflu- idic strategies to produce surfactant-DNA nanoparticles with a good control on their morphological properties. 2 Supramolecular structure: the case of lipid-DNA nanoparticles The architecture of simple viruses consists of the ge- nome encoded in nucleic acids, which are compacted and protected inside a protein shell called the capsid. Remarkably, the capsid alone [19] or the capsid with genome [20] can self-assemble in vitro from purified components. Nonviral DNA-based nanoparticles try to mimic this architecture. Likewise, they result from a self-assembly process, which is driven by a delicate bal- ance between weak (H bond, hydrophobicity, entropic effects) and strong (electrostatics, van der Waals forces) noncovalent interactions [21]. DNA is a negatively- charged polyelectrolyte and undergoes a coil-globule transition upon the addition of positively-charged agents, which can be synthetic polyelectrolytes, pep- tides, lipids or surfactants. This compaction process can be further enhanced by attractive interactions between positively-charged agents via hydrophobic forces as is the case with the alkyl chains of lipids and surfactants. Resultantly, the self-assembly of such DNA- based nanoparticles is driven both by electrostatics and by hydrophobic interactions, and it can give rise to a rich phase diagram. Lipids have played an important role in nonviral gene delivery because they are the main constituents of cell membranes. A lipid-based vector has thereby the ability to fuse with the membranes of host cells and to release efficiently its DNA. Lipids are organic molecules made up of a hydrophilic charged head and a hydrophobic alkyl tail [22]. When dispersed in water, they self-assem- ble into 4~5 nm-thick bilayers in such a way that the al- kyl tails are protected from the aqueous environment. At high volume fractions, the bilayers become stacked into a lamellar phase denoted La. More importantly, when cationic lipids are mixed with DNA, they form nanoparticles with local liquid-crystal order. Depend- ing on the shape of the lipid molecule, i.e., cylindrical or conical, we mostly observe complexed lamellar La C and complexed inverted hexagonal HII C phases [22]. The La C phase consists of alternating monolayers of DNA rods and lipid bilayers. In the HII C phase, DNA rods are coated by a lipid monolayer and arranged on a two-dimen- sional hexagonal lattice. Very interestingly, lipid-DNA nanoparticles in HII C phase transfer their DNA to cells much more efficiently than those in La C phase. Howev- er, cationic lipids are toxic to cells because they interact strongly with the negatively-charged membranes and disturb their biological functions. An alternative option is to use natural anionic lipids associated with DNA via multivalent cations [23]. The cations, in weak amounts, are intercalated between lipids and DNA [24], and the complexed lamellar and inverted hexagonal phases are recovered. The transfer efficiency of DNA is similar to that obtained with cationic lipids but the toxicity level is significantly lower. At large scale, lipid-DNA nanoparticles exhibit a certain degree of disorder. When cationic lipids and DNA are G. Tresset et al; Informacije Midem, Vol. 46, No. 4(2016), 183 – 189