Multiscale Approach for Tuning Communication among Chemical Oscillators Confined in Biomimetic Microcompartments.

ConspectusInspired by the biological world, new cross-border disciplines and technologies have emerged. Relevant examples include systems chemistry, which offers a bottom-up approach toward chemical complexity, and bio/chemical information and communication technology (bio/chemical ICT), which explores the conditions for propagating signals among individual microreactors separated by selectively permeable membranes. To fabricate specific arrays of microreactors, microfluidics has been demonstrated as an excellent method. In particular, droplet-based microfluidics is a powerful tool for encapsulating biological entities and chemical reagents in artificial microenvironments, mostly water-in-oil microdroplets. In these systems, the interfaces are liquid-liquid, and their physicochemical properties are key factors for tuning the coupling between molecular diffusion. Simple and double emulsions, where aqueous domains are in equilibrium with oil domains through boundary layers of amphiphilic molecules, are organized assemblies with high interfacial-area-to-volume ratios. These membranes can be engineered to obtain different surface charges, single- or multilayer stacking, and a variable degree of defects in molecular packing. Emulsions find application in many fields, including the food industry, pharmaceutics, and cosmetics. Furthermore, micro- and nanoemulsions can be used to model the propagation of chemical species through long distances, which is not only vital for cell signaling but also significant in molecular computing. Here we present in-depth research on the faceted world of solutions confined in restricted environments. In particular, we focused on the multiscale aspects of structure and dynamics from molecular to micro and macro levels. The Belousov-Zhabotinsky chemical reaction, known for its robustness and well-documented oscillatory behavior, was selected to represent a generic signal emitter/receiver confined within microenvironments separated by liquid-liquid interfaces. In this pulse generator, the temporal and spatial progressions are governed by periodic fluctuations in the concentration of chemical species, which act as activatory or inhibitory messengers over long distances. When organized into "colonies" or arrays, these micro-oscillators exhibit emergent dynamical behaviors at the population level. These behaviors can be finely tuned by manipulating the geometrical distribution of the oscillators and the properties of the interfaces at the nanoscale. By carefully selecting the membrane composition, it is possible to drive the system toward either in-phase, antiphase, or mixed synchronization regimes among individual oscillators, depending on messenger molecules. This relatively simple lab-scale model replicates some of the communication strategies commonly found in biological systems, particularly those based on the passive diffusion of chemical and electrical signals. It can help shed light on fundamental life processes and inspire new implementations in molecular computing and smart materials.

Impact of airborne iron oxide nanoparticles on Tillandsia usneoides as a model plant to assess pollution in heavy traffic areas.

Due to the increasing evidence of widespread sub-micron pollutants in the atmosphere, the impact of airborne nanoparticles is a subject of great relevance. In particular, the smallest particles are considered the most active and dangerous, having a higher surface/volume ratio. Here we tested the effect of iron oxide (Fe3O4) nanoparticles (IONPs) with different mean diameter and size distribution on the model plant Tillandsia usneoides. Strands were placed in home-built closed boxes and exposed to levels of airborne IONPs reported for the roadside air, i.e. in the order of 107 - 108 items m-2. Plant growth and other morpho-physiological parameters were monitored for two weeks, showing that exposure to IONPs significantly reduced the length increment of the treated strands with respect to controls. A dose-dependence of this impairing effect was found only for particles with mean size of a few tens of nanometers. These were also proved to be the most toxic at the highest concentration tested. The IONP-induced hamper in growth was correlated with altered concentration of macro- and micronutrients in the plant, while no significant variation in photosynthetic activity was detected in treated samples. Microscopy investigation showed that IONPs could adhere to the plant surface and were preferentially located on the trichome wings. Our results report, for the first time, evidence of the negative effects of airborne IONP pollution on plant health, thus raising concerns about related environmental risks. Future research should be devoted to other plant species and pollutants to assess the impact of airborne pollution on plants and devise suitable attenuation practices.

Growth, physiological parameters and DNA methylation in Spirodela polyrhiza (L.) Schleid exposed to PET micro-nanoplastic contaminated waters.

The effects of polyethylene terephthalate micro-nanoplastics (PET-MNPs) were tested on the model freshwater species Spirodela polyrhiza (L.) Schleid., with focus on possible particle-induced epigenetic effects (i.e. alteration of DNA methylation status). MNPs (size âˆ¼ 200-300 nm) were produced as water dispersions from PET bottles through repeated cycles of homogenization and used to prepare N-medium at two environmentally relevant concentrations (∼0.05 g L-1 and ∼0.1 g L-1 of MNPs). After 10 days of exposure, a reduction in fresh and dry weight was observed in treated plants, even if the average specific growth rate for both frond number and area was not altered. Impaired growth was coupled with a MNP-induced decrease of chlorophyll fluorescence parameters (i.e. ΨETo and Piabs, indicators of photochemical efficiency) and starch concentration, as well as with alterations in plant ionomic profile and oxidative status. The methylation-sensitive amplification polymorphism (MSAP) technique was used to assess possible changes in DNA methylation levels induced by plastic particles. The analysis showed unusual hypermethylation in 5'-CCGG sites that could be implicated in DNA protection from dangerous agents (i.e. reactive oxygen species) or in the formation of new epialleles. This work represents the first evidence of MNP-induced epigenetic modifications in the plant world.

Nanoformulations with exopolysaccharides from cyanobacteria: enhancing the efficacy of bioactive molecules in the Mediterranean fruit fly control.

The increasing demand for food has required intensive use of pesticides which are hazardous to the ecosystem. A valid alternative is represented by biopesticides; however, these molecules are often insoluble in water, and poorly bioavailable. Nanopesticides can be engineered to reach a selected target with controlled release of the active principle. In this work, capsaicin, an irritant alkaloid from hot chili peppers, and hydroxytyrosol, a phenolic compound obtained from extra-virgin olive oil by-products, were loaded into innovative nanocarriers. These were designed ad hoc combining exopolysaccharides from the cyanobacteria Neocyanospira capsulata, and a lipid component, i.e., egg phosphatidylcholine. The polysaccharide was chosen for chemical affinity with the chitin of insect exoskeleton, while the lipids were introduced to modulate the carrier rigidity. The newly formed nanosystems were characterized by physico-chemical techniques and tested for their possible use in pest control programs. The Mediterranean Fruit Fly Ceratitis capitata Wiedemann, 1824 (Diptera, Tephriditae), a pest of the Mediterranean Region causing high economic losses, was used as a model insect. We found that the nanoformulations nanocarriers prepared in this work, were able to increase the ovicidal effect of hydroxytyrosol. Moreover, the formulation encapsulating either hydroxytyrosol or capsaicin were able to reduce the number of females landing on treated apricots.

Tannins-lignin mixed nanoformulations for improving the potential of neem oil as fungicide agent.

Sustainability and circular economy are increasingly pushing for the search of natural materials to foster antiparasitic treatments, especially in the case of economically relevant agricultural cultivations, such as grapevine. In this work, we propose to deliver neem oil, a natural biopesticide loaded into novel nanovectors (nanocapsules) which were fabricated using a scalable procedure starting from Kraft lignin and grapeseed tannins. The obtained formulations were characterized in terms of size and Zeta potential, showing that almost all the nanocapsules had size in the suitable range for delivery purposes (mean diameter 150-300 nm), with low polydispersity and sufficient stability to ensure long shelf life. The target microorganisms were three reference fungal pathogens of grapevine (Botrytis cinerea, Phaeoacremonium minimum, Phaeomoniella chlamydospora), responsible for recurrent diseases on this crop: grey mold or berry rot by B. cinerea and diseases of grapevine wood within the Esca complex of diseases. Results showed that grapeseed tannins did not promote inhibitory effects, either alone or in combination with Kraft lignin. On the contrary, the efficacy of neem oil against P. minimum was boosted by more than 1-2 orders of magnitude and the parasite growth inhibition was higher with respect to a widely used commercial pesticide, while no additional activity was detected against P. chlamydospora and B. cinerea.

Fluorescent Labeling of Lignin Nanocapsules with Fluorol Yellow 088.

The microscopic visualization of nanoparticles in plants is crucial to elucidate the mechanisms of their uptake through the cell wall and plasma membrane and to localize the possible sites of their extracellular or intracellular accumulation. Lignin nanocarriers are polymeric hollow nanocapsules able to contain and transport several bioactive substances inside plant tissues. We describe here a method for the preparation of Fluorol Yellow 088-labeled lignin nanocapsules that allow their localization in plant organs and tissues by fluorescence microscopy.

Structuring and De-Structuring of Nanovectors from Algal Lipids: Simulated Digestion, Preliminary Antioxidant Capacity and In Vitro Tests.

Biocompatible nanocarriers can be obtained by lipid extraction from natural sources such as algal biomasses, which accumulate different lipid classes depending on the employed culture media. Lipid aggregates can be distinguished according to supramolecular architecture into lamellar and nonlamellar structures. This distinction is mainly influenced by the lipid class and molecular packing parameter, which determine the possible values of interfacial curvature and thus the supramolecular symmetries that can be obtained. The nanosystems prepared from bio-sources are able to self-assemble into different compartmentalized structures due to their complex composition. They also present the advantage of increased carrier-target biocompatibility and are suitable to encapsulate and vehiculate poorly water-soluble compounds, e.g., natural antioxidants. Their functional properties stem from the interplay of several parameters. Following previous work, here the functionality of two series of structurally distinct lipid nanocarriers, namely liposomes and cubosomes deriving from algal biomasses with different lipid composition, is characterized. In the view of their possible use as pharmaceutical or nutraceutical formulations, both types of nanovectors were loaded with three well-known antioxidants, i.e., curcumin, α-tocopherol and piperine, and their carrier efficacy was compared considering their different structures. Firstly, carrier stability in biorelevant conditions was assessed by simulating a gastrointestinal tract model. Then, by using an integrated chemical and pharmacological approach, the functionality in terms of encapsulation efficiency, cargo bioaccessibility and kinetics of antioxidant capacity by UV-Visible spectroscopy was evaluated. Subsequently, in vitro cytotoxicity and viability tests after administration to model cell lines were performed. As a consequence of this investigation, it is possible to conclude that nanovectors from algal lipids, i.e., cubosomes and liposomes, can be efficient delivery agents for lipophilic antioxidants, being able to preserve and enhance their activity toward different targets while promoting sustained release.

Plastic is in the air: Impact of micro-nanoplastics from airborne pollution on Tillandsia usneoides (L.) L. (Bromeliaceae) as a possible green sensor.

Due to the increasing evidence of widespread plastic pollution in the air, the impact on plants of airborne particles of polycarbonate (PC), polyethyleneterephthalate (PET), polyethylene (PE), and polyvinylchloride (PVC) was tested by administering pristine and aged airborne micro-nanoplastics (MNPs) to Tillandsia usneoides for two weeks. Here we showed that exposure to pristine MNPs, significantly reduced plant growth with respect to controls. Particularly, PVC almost halved plant development at the end of the treatment, while the other plastics exerted negative effects on growth only at the beginning of the exposure, with final stages comparable to those of controls. Plants exposed to aged MNPs showed significantly decreased growth at early stages with PC, later in the growth with PE, and even later with PET. Aged PVC did not exert a toxic effect on plants. When present, the plastic-mediated reduction in plant growth was coupled with a decrease in photosynthetic activity and alterations in the plant concentration of macro- and micronutrients. The plastic particles were showed to adhere to the plant surface and, preferentially, on the trichome wings. Our results reported, for the first time, evidence of negative effects of airborne plastic pollution on plant health, thus raising concerns for related environmental risks.

Synchronization scenarios induced by delayed communication in arrays of diffusively coupled autonomous chemical oscillators.

We study the impact of delayed feedbacks in the collective synchronization of ensembles of identical and autonomous micro-oscillators. To this aim, we consider linear arrays of Belousov-Zhabotinsky (BZ) oscillators confined in micro-compartmentalised systems, where the delayed feedback mimics natural lags that can arise due to the confinement properties and mechanisms driving the inter-oscillator communication. The micro-oscillator array is modeled as a set of Oregonator-like kinetics coupled via mass exchange of the chemical messengers. Changes in the synchronization patterns are explored by varying the delayed feedback introduced in the messenger species Br2. A direct transition from anti-phase to in-phase synchronization and back to the initial anti-phase scheme is observed by progressively increasing the time delay from zero to the value T0, which is the oscillation period characterising the system without any delayed coupling. The route from anti- to in-phase oscillations (and back) consists of regimes where windows of in-phase oscillations are periodically broken by anti-phase beats. Similarities between these phase transition dynamics and synchronization scenarios characterising the coordination of oscillatory limb movements are finally discussed.

Tip Streaming of a Lipid-Stabilized Double Emulsion Generated in a Microfluidic Channel.

Water/oil/water (w/o/w) double emulsions (DEs) are multicompartment structures which can be used in many technological applications and in fundamental studies as models of cell like microreactors or templates for other materials. Herein, we study the flow dynamics of water/oil/water double emulsions generated in a microfluidic device and stabilized with the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). We show that by varying the concentration of lipids in the oil phase (chloroform) or by modulating the viscosity of the aqueous continuous phase, the double emulsions under flow exhibit a rich dynamic behavior. An initial deformation of the double emulsions is followed by tube extraction at the rear end, relative to the flow direction, resulting in pinch off at the tube extremity by which small aqueous compartments are released. These compartments are phospholipid vesicles as deduced from fluorescence experiments. The overall process can thus be of help to shed light on the mechanical aspects of phenomena such as the budding and fusion in cell membranes.

Green and cost-effective synthesis of copper nanoparticles by extracts of non-edible and waste plant materials from Vaccinium species: Characterization and antimicrobial activity.

The aim of this work was the green synthesis of copper nanoparticles (Cu-NPs) using aqueous extracts of (i) bilberry (Vaccinium myrtillus L.) waste residues from the production of fruit juices and (ii) non-edible "false bilberry" fruits (Vaccinium uliginosum L. subsp. gaultherioides). Different cupric salts (CuCl2, Cu(CH3COO)2 and Cu(NO3)2) were used for the synthesis. The formation of stable nanoparticles (CuNPs) was assessed by transmission electron microscopy and the oxidation state of copper in these aggregates was followed by X-ray photoelectron spectroscopy. The polyphenol composition of the extracts was characterized, before and after the synthesis, using spectrophotometric methods (i.e. total soluble polyphenols and total monomeric anthocyanins) and high-performance liquid chromatography coupled with tandem mass spectrometry (i.e. individual anthocyanins). Polyphenol concentration in the extracts was found to decrease after the synthesis, indicating their active participation to the processes, which led to the formation of Cu-NPs. The antimicrobial activity of Cu-NPs, berry extracts, and cupric ion solutions were analysed by broth microdilution and time-kill assays, on prokaryotic and eukaryotic models. The antimicrobial activity of Cu-NPs, especially those derived from bilberry waste residues, appeared to be higher for both Gram-negative and Gram-positive bacteria, and for fungi, compared to the ones of its single components (cupric salts and berry extracts). Therefore, Cu-NPs from the green synthesis here proposed can be considered as a cost-effective sanitization tool with a wide spectrum of action.

Lipids from algal biomass provide new (nonlamellar) nanovectors with high carrier potentiality for natural antioxidants.

Lipid mesophases are lyotropic liquid crystalline systems which differ from liposomes and other globular aggregates in dilute regimes due to their inner ordering. It is known that natural lipids enable to obtain a rich variety of nanosystems and many of them have been proposed as delivery agents for bioactive compounds. Due to their packing parameters, several classes of lipids found in natural sources are able to self-assemble into nonlamellar structures. Among lipids occurring in plants and algae, triglycerides display this tendency. In the present study we examine new nanosystems built with lipids extracted from the marine microalga Nannochloropsis sp and their use as carriers for lipophilic antioxidants. The antioxidants studied, curcumin and tocopherol were encapsulated with high rate in the carriers. The physico-chemical characterization of plain and loaded vectors showed their structure and localization site, as well as the structure-functionality relationship related to potential drug delivery. The results show that the cargo molecules play an active role in driving the interactions which characterize the overall structure of the aggregates. The systems studied showed several coexisting mesophases, the most predominant structure being of cubic symmetry.

Microfluidic compartmentalization of diffusively coupled oscillators in multisomes induces a novel synchronization scenario.

Stable cell-like multisomes encapsulating the chemical oscillator Belousov-Zhabotinsky were engineered and organized in a linear network of diffusively-coupled chemical oscillators by using microfluidics. The multi-compartmentalization and the spatial configuration resulted in a new global synchronization scenario. After an initial induction interval, all the oscillators started to pulsate in phase with a halved period with respect to the natural one.

A new method for the direct tracking of in vivo lignin nanocapsules in Eragrostis tef (Poaceae) tissues.

Environmental concerns have driven scientists to research new eco-friendly approaches for the preparation of nanosystems. For this purpose, novel bio-polymers have been selected. Among these, one of the most promising is lignin, which is biodegradable and biocompatible. Additionally, lignin is one of the main by-products of the paper industry and can be re-used in nanosystems building. Lignin-based nanosystems could be used in agriculture, to improve the uptake of bioactive compounds, thus avoiding soil pollution. However, the mechanism of penetration in the plant and the route of transportation within the internal plant tissues are unknown and need to be clearly elucidated. Here we present a method of lignin nanocapsules staining and tracking by fluorochrome: Fluoral Yellow 088, which is a well-suited dye for the tracking of lipids and other oil phases. Two different applications were applied: in the first one fourteen-day plants were soaked with fluorescent nanocapsules (fNCs) pure solution and in the second one, Eragrostis tef plants were laid down on blotting paper and soaked with diluted fNCs solution. Wetting the roots of Teff plantlets with the pure fNCs solution resulted in the most efficient way of nanocapsule entrance. The dyeing of lignin nanocapsules allowed us to track them in Eragrostis tef plant tissues through microscopic observations. In particular, fNCs were proven to be able to permeate roots, reaching xylem vessels where, through water pressure, they reached the leaf.

Membrane Structure Drives Synchronization Patterns in Arrays of Diffusively Coupled Self-Oscillating Droplets.

Networks of diffusively coupled inorganic oscillators, confined in nano- and microcompartments, are effective for predicting and understanding the global dynamics of those systems where the diffusion of activatory or inhibitory signals regulates the communication among different individuals. By taking advantage of a microfluidic device, we study the dynamics of arrays of diffusively coupled Belousov-Zhabotinsky (BZ) oscillators encapsulated in water-in-oil single emulsions. New synchronization patterns are induced and controlled by modulating the structural and chemical properties of the phospholipid-based biomimetic membranes via the introduction of specific dopants. Doping molecules do not alter the membrane basic backbone, but modify the lamellarity (and, in turn, the permeability) or interact chemically with the reaction intermediates. A transition from two-period clusters showing 1:2 period-locking to one-period antiphase synchronization is observed by decreasing the membrane lamellarity. An unsynchronized scenario is found when the dopant is able to interfere with chemical communication by reacting with the chemical messengers.

Exploring the water/oil/water interface of phospholipid stabilized double emulsions by micro-focusing synchrotron SAXS.

Surfactant stabilized water/oil/water (w/o/w) double emulsions have received much attention in the last years motivated by their wide applications. Among double emulsions, those stabilized by phospholipids present special interest for their imitation of artificial cells, allowing the study of the effect of confining chemical reactions in biomimetic environments. Upon evaporation of the oil shell, phospholipid stabilized double emulsions can also serve as templates for giant vesicles. In this context, general assumptions have been made on the self-assembly and structural organization/arrangement of amphiphilic molecules, at the aqueous/oil liquid interface. However, to the best of our knowledge, no detailed evidence of the interfacial structuring have been reported. In this paper, w/o/w double emulsions formulated using the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a mixture of chloroform and cyclohexane as the oil phase were produced using a microfluidic device. To obtain information on the phospholipid arrangement, the w/o/w interface was investigated by spatially resolved micro-focusing SAXS. We observed that (i) the basic units forming both the w/o and o/w interfaces were oil-swollen DMPC bilayers, arranged into a substantially disordered shell of ∼45 µm thickness surrounding the internal oil phase; (ii) the evaporation process was slow, i.e. in the order of one hour at 50 °C and (iii) oil evaporation led to a shrinkage of the interfacial shell, but not to an increase of the ordering of the lipid bilayers. Interestingly, no stacked DMPC bilayers were observed during the evaporation process, as shown by the absence of Bragg's peaks in the SAXS intensity profiles.

Structural Characterization of Self-Assembling Hybrid Nanoparticles for Bisphosphonate Delivery in Tumors.

Hybrid self-assembling nanoparticles (hsaNPs) encapsulating bisphosphonates (BPs) recently showed very promising results in preclinic experiments for the treatment of brain tumor. However, the poor knowledge on the architecture of hybrid nanovectors is certainly one of the main reasons hampering further clinical and industrial development of these technologies. Here we propose to combine different techniques, that is, small angle neutron scattering (SANS) and X-ray Sscattering (SAXS), with cryo-electron transmission microscopy (cryo-TEM) to study the architecture of the final hsaNPs as well as of the four components before the assembling process. Data analysis based on SANS and SAXS experiments suggested a multiple compartment architecture of the final product, consisting of two bilayers sourrounding a core. Structures consisting of two shells surrounding an internal core were also observed in the cryo-TEM analysis. Such high resolution insight, also combined with size distribution and zeta potential of the NPs, provides exhaustive characterization of hsaNPs encapsulating BPs, and it is aimed at supporting further their clinical and industrial development.

Lipid-Stabilized Water-Oil Interfaces Studied by Microfocusing Small-Angle X-ray Scattering.

Water-in-oil (w/o) simple emulsions are dispersed microconfined systems that find applications in many areas of advanced materials and biotechnology, such as the food industry, drug delivery, and cosmetics, to name but a few. In these systems, the structural and chemical properties of the boundary layer at the w/o interface are of paramount importance in determining functionality and stability. Recently, microfluidic methods have emerged as a valuable tool for fabricating monodisperse emulsion droplets. Because of the intrinsic flexibility of microfluidics, different interfaces can be obtained, and general principles governing their stability are needed to guide the experimental approach. Herein, we investigate the structural characteristics of the region encompassing the liquid/liquid (L/L) interface of w/o emulsions generated by a microfluidic device in the presence of phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and other intercalating amphiphiles (dopants) using microfocused small-angle X-rays scattering (µ-SAXS). We show that phospholipids provide a stable and versatile boundary film of ∼100 µm whose basic units are swollen and uncorrelated DMPC bilayers. The internal arrangement of this interfacial film can be tuned by adding molecules with a different packing parameter, such as cholesterol, which is able to increase the stiffness of the lipid membranes and trigger interbilayer correlation.

Chemical communication and dynamics of droplet emulsions in networks of Belousov-Zhabotinsky micro-oscillators produced by microfluidics.

Chemical communication leading to synchronization and collective behaviour of dynamic elements, such as cell colonies, is a widespread phenomenon with biological, physical and chemical importance. Such synchronization between elements proceeds via chemical communication by emmision, interdiffusion and reception of specific messenger molecules. On a lab scale, these phenomena can be modeled by encapsulating an oscillating chemical reaction, which serves as a signal (information) sender/receiver element, inside microcompartments such as droplet emulsions, liposomes and polymersomes. Droplets can thus be regarded as single units, able to generate chemical messengers that diffuse in the environment and hence can interact with other compartments. The Belousov-Zhabotinsky (BZ) reaction is a well-known chemical oscillator largely used as a model for complex nonlinear phenomena, including chemical, physical and biological examples. When the BZ-reaction is encapsulated inside microcompartments, its chemical intermediates can serve as messengers by diffusing among different microcompartments, to trigger specific reactions leading to a collective behavior between the elements. The geometry and constitution of the diffusion pathways play an important role in governing the collective behaviour of the system. In this context, microfluidics is not only a versatile tool for mastering the encapsulation process of the BZ-reaction in monodisperse microcompartments, but also for creating geometries and networks with well defined boundaries. The individual compartments can be engineered with selected properties using different surfactants in the case of simple emulsions, or with specific membrane properties in the case of liposomes. Furthermore, it enables the arrangement of these microcompartments in various geometric configurations, where the diffusive coupling pathways between individual compartments are both spatially and chemically well-defined. In this tutorial paper, we review a number of articles reporting various approaches to generate networks of compartmentalized Belousov-Zhabotinsky (BZ) chemical oscillators using microfluidics. In contrast to biological cellular networks, the dynamical characteristics of the BZ-reaction is well-known and, when confined in microcompartments arranged in different configurations with a pure interdiffusive coupling, these communicative microreactors can serve to mimic various types of bio-physical networks, aiding to comprehend the concept of chemical communication.

Complexation of short ds RNA/DNA oligonucleotides with Gemini micelles: a time resolved SAXS and computational study.

Gene therapy is based on nucleic acid delivery to pathogenic cells in order to modulate their gene expression. The most used non viral vectors are lipid-based nanoaggregates, which are safer than viral carriers and have been shown to assemble easily with both DNA and RNA. However, the transfection efficiency of non viral carriers still needs to be improved before intensive practise in clinical trials can be implemented. For this purpose, the in depth characterization of the complexes formed by nucleic acids and their transporters is of great relevance. In particular, information on the structure and assembly mechanism can be useful to improve our general knowledge of these artificial transfection agents. In this paper, the complexation mechanism of short interfering RNA and DNA molecules (siRNA and siDNA, respectively) with cationic micelles is investigated by combining small angle X-ray scattering experiments and molecular dynamics simulations. Micelles were obtained by Gemini surfactants with different spacer lengths (12-3-12, 12-6-12). The siRNA and siDNA used were double strand molecules characterized by the same length and homologous sequence, in order to perform a close comparison. We showed that complexes appear in solution immediately after mixing and, therefore, the investigation of complex formation requires fast experimental techniques, such as time resolved synchrotron SAXS (Tr-SAXS). The obtained systems had internal arrangement constituted by layers of squeezed micelles alternating the nucleic acids. Both SAXS and MD analyses allowed us to evaluate the mean size of complexes in the range of a few nanometers, with looser and less ordered stacking for the DNA containing aggregates.