Employment of Cationic Solid-Lipid Nanoparticles as RNA Carriers

Moreover, we utilized the sea urchin as a model system to test their applicability on a living organism. To evaluate cationic SLN ability to complex t...
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Bioconjugate Chem. 2007, 18, 302−308

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ARTICLES Employment of Cationic Solid-Lipid Nanoparticles as RNA Carriers Giovanna Montana,†,⊥ Maria L. Bondı`,‡,⊥ Rita Carrotta,§,4 Pasquale Picone,† Emanuela F. Craparo,| Pier L. San Biagio,§ Gaetano Giammona,| and Marta Di Carlo*,† Istituto di Biomedicina e Immunologia Molecolare, Istituto per lo Studio dei Materiali Nanostrutturati, sezione di Palermo, and Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via Ugo La Malfa, 153, 90146 Palermo, and Dipartimento di Chimica e Tecnologie Farmaceutiche, Universita` di Palermo, via Archirafi, 32, 90123 Palermo, Italy. Received May 5, 2006; Revised Manuscript Received July 27, 2006

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. In this article, the suitability of cationically modified solid-lipid nanoparticles (SLN) as a nonviral vector for gene delivery was investigated, in order to obtain stable materials able to condense RNA. Cationic SLN were produced by microemulsion using Compritol ATO 888 as matrix lipid, Pluronic F68 as tenside, and dimethyldioctadecylammonium bromide (DDAB) as cationic lipid. The resulting particles were approximately 100 nm in size and showed a highly positive surface charge (+41 mV) in water. Size and shape were further characterized by scanning electron microscopy (SEM) measurements. Moreover, we utilized the sea urchin as a model system to test their applicability on a living organism. To evaluate cationic SLN ability to complex the in vitro transcribed Paracentrotus liVidus bep3 RNA, we utilized both light scattering and gel mobility experiments, and protection by nuclease degradation was also investigated. By microinjection experiment, we demonstrated that the nanoparticles do not inference with the viability of the P. liVidus embryo and the complex nanoparticles-bep3 permits movement of the RNA during its localization in the egg, suggesting that it could be a suitable system for gene delivery. Taken together, all these results indicate that the cationic SNL are a good RNA carrier for gene transfer system and the sea urchin a simple and versatile candidate to test biological properties of nanotechnology devices.

INTRODUCTION Gene delivery is an area of considerable current interest for molecular medicine. DNA and proteins have been extensively investigated for their application in nanotechnology as a possible approach to gene therapy (1, 2). Minor attention has been given to other important biological molecules such as RNA until now. In nature, RNA exhibits a single-stranded conformation, but it is versatile enough to form, through specific complementary sequences, stems, loops, and hairpins, providing elements of adaptability to nanotechnology that is based on a double-helical structure. The use of antisense or sense RNA is a specific and powerful tool for selectively modulating the expression of a gene target to identify loss-of-function or gain-of-function phenotypes (3-8). The use of RNA interference (RNAi), based on gene-silencing of key molecules involved in many diseases, has been an improvement in this technology (9). All these kinds of synthetic RNAs are generally introduced into the cells by electroporation and/or microinjection, but their poor stability is often the cause of failure. Another technology to introduce * To whom correspondence may be addressed. E-mail: [email protected]. Tel. ++39 0916809538; fax ++39 0916809548. † Istituto di Biomedicina e Immunologia Molecolare, Consiglio Nazionale delle Ricerche. ‡ Istituto per lo Studio dei Materiali Nanostrutturati, sezione di Palermo, Consiglio Nazionale delle Ricerche. § Istituto di Biofisica, Consiglio Nazionale delle Ricerche. 4 Present address: Dipartimento di Scienze Fisiche ed Astronomiche, Universita` di Palermo, via Archirafi 36, 90138 Palermo. | Universita ` di Palermo. ⊥ These authors contributed equally to this work.

RNA molecules employs viral vectors, but undesirable effects can occur (10, 11). In the past decade, many studies have shown that nonviral vectors, as cationic liposomes and polycations, are valid tools for delivering nucleic acids to specific cell types both to inhibit some undesirable gene expression or to express therapeutic proteins (12, 13). The use of nonviral vectors permits one to avoid some risk factors such as pathogenesis, immunogenicity, and so forth(14-16). Among them, cationic solid-lipid nanoparticles (SLN) as a carrier system for the delivery of genetic materials may offer several technological advantages (17). These include excellent storage stability, relatively easy production without the use of any organic solvent, the possibility of steam sterilization and lyophilization, and large-scale production utilizing substances that are approved for use in pharmaceutical human applications (18-22). These particles consist of a lipid core bearing cationic lipids on its surface. The aim of this study was to prepare and utilize cationically modified SLN as specific RNA carriers and to evaluate their potential as a nonviral vector for gene delivery. Moreover, we have utilized, as a living organism, the sea urchin Paracentrotus liVidus and its bep3 maternal messenger RNA (23, 24). Among the classical model systems, the sea urchin provides a powerful model for a great variety of studies in molecular and cell biology and biochemistry. Moreover, the sea urchin occupies a key phylogenetic position as the only nonchordate detereustomes, and the results obtained on this embryo can be extrapolated and compared to those of higher eukaryotes. Eggs and embryos are transparent, allowing direct observation of cell division and movement, and specific techniques permit us to visualize the presence of exogenous nucleic acids inside the cells. Further-

10.1021/bc0601166 CCC: $37.00 © 2007 American Chemical Society Published on Web 01/25/2007

SLN as Vector for RNA Delivery

more, bep3 RNA codifies for a cell surface protein, localized after fertilization at the animal pole of the Paracentrotus liVidus egg by moving along cytoskeleton elements (24, 25). Thus, this RNA represents a good model to study the delivery of nucleic acids. Interestingly, it has been demonstrated that bep3 3′UTR, necessary for its localization, could fold in secondary structures; that is a required element for nanotechnology (26). The ability to form complexes between SLN and bep3 and the effect on its mobility have been tested.

EXPERIMENTAL PROCEDURES Materials. Compritol ATO 888 is a mixture of approximately 15% mono-, 50% di-, and 35% triglycerides of behenic acid (C22). The melting point lies between 69 and 74 °C. It was a gift from Gattefosse´ (D-Weil am Rhein, Germany). Pluronic F68 and the cationic surfactant dimethyldioctadecylammonium bromide (DDAB) were purchased from Sigma-Aldrich. Preparation of Solid-Lipid Nanoparticles. The SLN were prepared from a warm oil-in-water (o/w) microemulsion by using Compritol ATO 888 and DDAB as the lipid matrix. Briefly, 0.273 mmol of Compritol were heated to 10 °C above its melting point and mixed with 2.5 mL of a hot aqueous solution of Pluronic F68 (0.158 mmol) and DDAB (1.45 mmol) to form a clear microemulsion, under mechanical stirring. Then, cationic nanoparticles were obtained by dispersing under mechanical stirring at 1000 rpm the warm o/w microemulsion in cold water (2-3 °C) (organic/aqueous volume ratio equal to 1:10). The obtained cationic nanoparticles were purified by dialysis using a Visking Tubing Dialysis 18/32′′ (with a molecular weight cutoff of 12 000-14 000 Da). Then, cationic nanoparticles were freeze-dried using a Modulo freeze-dryer (Labconco Corporation, Missouri). To label SLN, fluorescein free acid was added to the lipid phase during preparation of the microemulsion. In Vitro Synthesis of the Transcript. To synthesize bep3 RNA, the plasmid containing this clone was linearized by digestion at the Xho I site (24). Transcription by T7 polymerase was carried out utilizing the DIG-RNA labeling kit and DIGUTP as the substrate according to the manufacturer’s instructions in the kit (Roche). The DIG-labeled RNA probe was detected by immunoassay using an anti-digoxigenin alkaline phosphatase conjugate (anti DIG-AP) according to the manufacturer’s instructions (Roche). Preparation of Cationic SLN-RNA Complexes and RNA Retardation Assay. To four equal aliquots of a bep3 DIG-RNA (50 ngr) solution in 5 mM Hepes, increasing amounts of nanoparticle suspensions were added to obtain the final w/w ratios of 50:1, 100:1, 200:1, and 400:1. In order to evaluate the effect of the preparation method on the nature of the obtained complexes, these were prepared also by adding the RNA solutions to the cationic SLN suspensions. All the cationic SLN-RNA complexes prepared by using both methods were characterized in terms of mean size, ζ potential, and electrophoretic mobility, and the same results were achieved. After 30 min incubation at room temperature, RNA binding was studied by assaying for agarose gel retardation. Samples were electrophoresed through a 1% agarose gel at 80 V for 1 h. RNA was visualized by a UV transluminator using ethidium bromide staining. Size and ζ Potential Measurements. Particle or complex size was analyzed by photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instrument, Herrenberg, Germany) which utilizes the noninvasive back-scattering (NIBS) technique. PCS gives information about the mean diameter of the bulk population (so-called z-average) and the width of distribution via the polydispersity index (PI). Samples were appropriately diluted with filtered (0.2 µm) twice-distilled water,

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and the readings were carried at a 173° angle with respect to the incident beam. The reported values were the averages of three measurements. The surface charge was determined using the same equipment. The ζ potential value was measured using principles of the laser Doppler velocimetry and phase analysis light scattering (M3PALS technique). Samples were dispersed in filtered (0.2 µm) twice-distilled water and analyzed in triplicate. Light Scattering Measurements. A water dispersion of SLN at a final concentration of c1.2 mg/mL was filtered via a 0.45 µm filter directly into a cylindrical quartz cuvette. To study the interaction between SLN and bep3 RNA, a small aliquot of RNA solution (about 25 ng) was added in situ into the cuvette containing the filtered SLN suspension. A Brookhaven Instrument BI200-SM goniometer, equipped with a 100 mW Ar laser tuned at λ0 ) 488 nm, was used in a temperature-controlled setup, as reported elsewhere (27). Simultaneous measurement of the intensity autocorrelation function and static light scattered at 90° proved the stability of the SLN suspension for 60 min. Immediately after mixing the RNA into the suspension of nanoparticles, the interaction between SLN and bep3 RNA was monitored by time-resolved light scattering experiments. Both the intensity autocorrelation function measured at 90° and static light scattered at 90°, averaged for 3 min, were measured independently to follow the process at T ) 25 °C. From the measured intensity autocorrelation functions, g2(t), we calculated the electric field autocorrelation functions, g1(t), considering the Siegert relation: g1(t) ) xg2(t) - 1 (28). The autocorrelation functions g1(t) were analyzed using a smoothing constrained regularization method, in order to obtain the distribution P(D) of the apparent diffusion coefficients D, according to the fact that g1(t) ) ∫P(D)e-Dq2t dD, where q is the scattering vector defined as q ) 4πn/λ0 sin θ/2, with n the medium refractive index and θ the scattering angle (29). By using the Stokes Einstein relationship, D ) KT/3πηDh (k is the Boltzmann’s constant, T the absolute temperature, η the solvent viscosity, and Dh the z-averaged hydrodynamic diameter), we obtain the distribution P(Dh) of the z-averaged hydrodynamic diameters of the species in suspension. The mean hydrodynamic diameter relative to a restricted interval of the distribution was estimated by calculating the harmonic average of the hydrodynamic diameter in the region of interest. Absolute scale for the scattered intensity was obtained by normalizing data to the toluene Rayleigh ratio at 488 nm, taken 39.6 × 10-6 cm-1. Scanning Electron Microscopy (SEM). A scanning electron microscope (SEM) (XL-30 Philips, The Netherlands) was used to evaluate both size and morphology of cationic SLN and SLN-RNA complexes obtained by using a w/w ratio of 200: 1. Before the SEM analysis, the samples were coated under an argon atmosphere with a 10-nm gold thickness. The samples were stuck onto aluminum stubs, which were then fixed into a sample holder and placed in the vacuum chamber of the microscope and observed under low vacuum (10-3 Torr). RNAse Degradation Assay. To evaluate the nuclease activity, the complex between cationic SLN-RNA obtained at a 200:1 w/w ratio was chosen, and the incubation was continued in the presence of 0.1, 1, and 10 u of RNAse T1 (Roche) or 0.1, 1, and 10 u of RNAse A (Roche) for an additional 30 min at room temperature. Electrophoresis of the RNA nanoparticle complex was carried out in 1% agarose gel at 80 V for 1 h. The gel was visualized by a UV transilluminator. Microinjection in Sea Urchin Eggs. Dejellied P. liVidus unfertilized eggs were microinjected as described by McMahon (30). When we microinjected the nanoparticles alone, 200 of the eggs were fertilized and left to develop for 48 h (pluteus stage). When we microinjected the complex SNL-bep3 in the 200:1 w/w ratio, before and after fertilization the eggs were

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Figure 1. ζ potential values of cationic SLN-RNA complexes at different weight ratios, measured in twice-distilled water after 30 min of incubation.

fixed in 4% paraformaldeide. The presence and movement of exogenous RNA were detected by immunoassay using an antidigoxigenin alkaline phosphatase conjugate (anti-Dig-AP) according to the manufacturer’s instructions of the DIG Nucleic Acid Detection kit (Roche). Viability Assay. Cell viability was determined using MTS assay according to manufacturer’s instructions (PROMEGA). This assay uses the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt (MTS), and the electron coupling reagent, phenazine methosulfate (PMS). MTS is chemically reduced by cells into formazan, which is soluble in culture medium. Briefly, MTS was added to embryos at 48 h of development (pluteus stage), to a final concentration of 0.8 mg/mL, and incubated for 5 h. The measurement of the absorbance of the formazan was carried out using a spectrophotometer at 495 nm.

RESULTS Production of Cationic SLN. The aim of this study was to prepare cationic nanoparticles consisting of Compritol, dimethyldioactadecylammonium bromide (DDAB), and Pluronic F68, to characterize them in water suspension (particle size and surface charge), and to study their interaction process with RNA. The SLN formulation was produced, as previously described, from a warm oil-in-water (o/w) microemulsion. In order to obtain a positive surface charge, a cationic lipid/surfactant DDAB was employed. The characterization of particle size by photon correlation spectroscopy (PCS) revealed that the average particle size of these particles was 100 nm (PI ) 0.215). The surface charge value was highly positive (+41 mV), demonstrating the successful incorporation of DDAB onto the nanoparticle surface. The formulation was stored in the dark and at room temperature. It showed excellent storage stability, with the average particle diameter changing only by a few nanometers during 180 days (data not shown). The binding of RNA with cationic SLN was evaluated by measurements of the ζ potentials of complexes at different cationic SLN-RNA weight ratios. The trend of the ζ-potential measurements of these complexes in water as a function of SLN-RNA weight ratios is illustrated in Figure 1. It is shown that, by increasing the used weight amount of nanoparticles in the complex formation, ζ potential values increased, starting from a value of -17 mV for naked RNA, up to 20 mV, passing through a neutral condition at a SLN-RNA weight ratio of 200:1. These results confirm the good capacity of these cationic nanoparticles to complex RNA and neutralize its anionic charge. SLNs are Able to Complex RNA. To investigate if cationic SLN can be applied in RNA nanotechnology, we followed the aggregation kinetics of the two components by light scattering. For this aim, we utilized Paracentrotus liVidus bep3 RNA whose characteristics have been described before (24-26). The static light scattered and the intensity autocorrelation functions both

Figure 2. (A) Static light scattered at 90° after mixing of SNL and bep3 RNA particles. The continuous line represents the best fit by using an exponential function. (B) Distribution functions of the hydrodynamic diameter, P(Dh), at four different times during the kinetic process, at t ) 0 without RNA (continuous black line), at t ) 25 min after mixing (dashed black line), at t ) 35 min after mixing (red line), and at t ) 155 min after mixing (blue line). The distribution functions were obtained by using a smoothing constrained regularization method to analyze the field autocorrelation functions (g1(t) data and resulting fits are shown in the inset). (C) Time dependence of the z-average hydrodynamic diameter corresponding to the region 40 nm < Dh < 400 nm.

at 90° have been measured simultaneously and independently in order to follow the interaction process between SLN and bep3 RNA. Figure 2A shows an appreciable, though noisy, Rayleigh ratio increase. The data fit, by using an exponential growth function, gives an estimate of the characteristic interaction time, τ, of 16 min and of the process saturation time on the order of 100 min. Figure 2B reports on the distribution function of hydrodynamic diameters in the range 40-400 nm at different times during the interaction process obtained by the analysis of the field autocorrelation function data. From the field autocorrelation functions (data shown in the inset), one can notice a decay in

SLN as Vector for RNA Delivery

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Figure 3. Binding of sense RNA to cationic nanoparticles. 50 ng of RNA was incubated with increasing amounts of cationic nanoparticles. Uncomplexed bep3 RNA (1), cationic SLN-bep3 complexes in a w/w ratio of 50:1 (2), 100:1 (3), 200:1 (4), and 400:1 (5). The arrow indicates the uncomplexed RNA; the arrowhead indicates the RNA trapped in the well. Figure 5. bep3 RNA-SNL complex is protected by RNAse enzymatic degradation. The RNA-cationic nanoparticle complexes were incubated without (C) or with RNA T1 A or with RNAse A at different amounts (0.1; 1; 10 units). Uncomplexed RNA incubates or not (C) with RNAse A.

Figure 4. SEM images of (A) cationic SLN and (B) group of representative cationic SLN-RNA complexes obtained at a w/w ratio of 200:1. The arrows indicate the complexes; the arrowhead indicates the free nanoparticles.

the time interval 0.1-1 ms, corresponding to particles with hydrodynamic diameters between 40 and 400 nm, that is to SLN at the beginning (t ) 0) and to SLN-RNA complexes later on during their formation. A faster decay due to smaller particles with hydrodynamic dimensions of about 5 nm was noticed in the autocorrelation functions (data not shown). Such a contribution, constant during the entire process, suggested the probable presence in solution of small lipid aggregates not interacting with RNA. Figure 2C shows the values of the mean hydrodynamic diameter calculated for the band of particles with hydrodynamic dimensions between 40 and 400 nm. The increase of the hydrodynamic diameter for the SLN-RNA particles has been fitted by using a function expressing an exponential growth giving a characteristic time, τ, of 13 min, a saturation time process of about 100 min, and an average diameter increase, ∆Dh, of about 80 nm. Bep3 RNA Mobility is Retarded by SNL. In order to evaluate the ability of cationic SLN to bind bep3 RNA, the RNA electrophoretic mobility shift within an agarose gel was investigated. Efficient complexation of bep3 by cationic SNL led to immobilization and prevention of ethidium bromide intercalation with RNA. In vitro transcribed bep3 RNA was incubated with increasing amounts of cationic SLN for 30 min. 50 to 100 weight equivalent were sufficient to bind most of RNA, while 200 to 400 completely immobilized RNA. As shown in Figure 3, bep3 is able to form complexes with

Figure 6. SNL microinjection into Paracentrotus liVidus unfertilized eggs. Cationic nanoparticles were microinjected into unfertilized eggs and left to develop for 48 h until pluteus stage (B) as control embryos (A). (C) Vitality of microinjected embryos compared with control embryos. On the right, the percentage of survived embryos is indicated. Standard deviation is 3.

nanoparticles in a dose-dependent manner. As nanoparticle concentration increased, the RNA exhibited decreased migration

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Figure 7. Localization of bep3 RNA in Paracentrotus liVidus unfertilized (A) or fertilized (B) eggs after microinjection with cationic nanoparticles. (C) Untreated P. liVidus egg as control. (D) P. liVidus egg after incubation with fluorescent SNL. (E) P. liVidus egg after incubation with fluorescent SNL complexed to RNA and stained with anti-DIG.

strength, and when the complex was too large, it did not enter into the gel, staying trapped in the well. This behavior was probably due both to the increased mass and to the modification of the RNA’s electric charges. Cationic SLN, indeed, spontaneously reacted with the negatively charged RNA molecules by the formation of ion pairs between the hydrophobic cations and the negatively charged phosphate groups of the nucleic acid chain, forming complexes with 100% RNA molecules involved in the reaction, in a selfassembling manner. When all the RNA negative charges are neutralized by the positive charges of the SLN surface, the RNA is no longer able to migrate. Analysis of Cationic SLN and SLN-RNA Complexes by SEM. To examine the size and the shape of the produced cationic SLN and SLN-RNA complexes obtained at a w/w ratio equal to 200:1, SEM analysis was performed on the sample. Figure 4A,B shows cationic SLNs and SLN-RNA complexes, respectively. By SEM images, we detected that the nanoparticles alone are spherical and have a diameter of approximately 100 nm (Figure 4A). Figure 4B shows an overview of SLN-RNA complexes; many coupled nanoparticles, with diameters between 200 and 300 nm, are present in the sample, indicating that the complex with RNA probably involves two or three nanoparticles. Moreover, free cationic SLN were found into the same sample (Figure 4B). SLN Protect RNA by Enzymatic Degradation. A peculiar problem, when RNA molecules are in vitro or in vivo transferred in any type of cells, is their susceptibility toward nuclease activity. Thus, nuclease could destroy an RNA molecule before it is able to arrive to the target site. Exogenous RNA is, indeed, unstable in biological fluid or intracellular environments. To test the protection ability of the nanoparticles toward RNA by nuclease attack, we have incubated the bep3-SNL complex with increasing amounts until excessive concentrations of two types of ribonucleases RNAse A and RNAse T1 are reached. The difference between these two endonucleases consists of the ability of T1 to digest only unprotected RNA with respect to the ability of A to digest any RNA. After separate digestion with RNAse A and T1, the complexes underwent to electrophoresis. Results, shown in Figure 5, suggest that nanoparticles protect bep3 RNA by RNAse T1 enzymatic degradation, while

it remains exposed to digestion by RNAse A only when a huge excess, in any case not present in the cellular environment, was utilized. Microinjection of SLN in Sea Urchin Eggs. To investigate the eventual SLN toxicity on a living organism and if they are harmful for RNA delivery, we utilized a classical model system such as the sea urchin Paracentrotus liVidus. Different amounts of SLN were injected into P. liVidus eggs. After fertilization, the embryos were left to develop for 48 h until the control arrived at the so-called pluteus stage (a primordial larva). Both microinjected embryos and the control showed the correct morphological aspect of this sea urchin embryonal stage, demonstrating that SLN are not toxic for this living organism (Figure 6A,B). Moreover, the viability of microinjected P. liVidus embryos was monitored by MTS assay (Figure 6C). Successively, to investigate the RNA delivery ability of SLN, the complex SLN-bep3 RNA in unfertilized P. liVidus eggs was microinjected and its localization was observed before and after fertilization. The RNA movement was detected after incubation with anti-DiG, an antibody able to recognize the UTP-DIG conjugate of the in vitro transcribed bep3. As shown in Figure 7A, before fertilization, bep3 RNA is distributed throughout the cytoplasm. After fertilization (see Figure 7B), instead, staining both under the cellular membrane and particularly in the subcortical region around the nucleus was detected, indicating that the SLN have not interfered with the normal movement of this RNA (24). Moreover, the possibility of SLN passing through the biological cellular membrane was tested. Fluorescein-loaded SLN, alone or complexed with RNA, were incubated with sea urchin eggs and after 16 h were observed with a fluorescence microscope. As shown in Figure 7D, the eggs are completely fluorescent, in particular forming a layer around the egg and indicating the perfect ability of SLN to diffuse through the cellular membrane. Finally, fluorescein-loaded SNL complexed to bep3 were incubated with P. liVidus eggs and the ability of the RNA to enter the SNL by diffusion was detected after incubation with anti-DIG (Figure 7E).

DISCUSSION The emergent field of nanotechnology generally involves the characterization, modification, and assembly of organized

SLN as Vector for RNA Delivery

Figure 8. Schematic representation of the interaction between RNA and cationic nanoparticles in the complex.

materials on the nanoscale level. The application of nanotechnology in life sciences is already having an impact on diagnostics and gene therapy in the fight against cancer and genetic disorders. Since biological macromolecules have intrinsic features at the nanometer scale, they have the potential characteristics for formation of nanostructures or nanodevices. RNA is a particular candidate for such application and can exhibit the right versatility to form complexes with nonviral vectors such as cationic nanoparticles. The use of this kind of vector can help genetic material cross the plasmatic membrane and enter the cytoplasm of a living cell and could avoid immunogenic and cytotoxic problems. In this paper, we describe the production of a new type of cationic nanoparticle, and we test both their ability to form a complex and protect RNA and their toxicity and biocompatible properties on a living organism such as the sea urchin. Cationic SLN have been used to incorporate RNA, and the self-assembled RNA nanostructures are formed with dimensions that fit well within the range of nanotechnology. As measured by dynamic light scattering, the average hydrodynamic diameter of cationic SLN with RNA in a water solution can be estimated at about 100 nm. After 100 min of mixing, the increase of the average hydrodynamic diameter to about 180 nm confirms the formation of SLN-RNA complexes. From these data, which can only give averaged information, it is hard to assume any kind of model for the complex. However, an indication that very few SLN (two or three) are involved in the complex can be ruled out (Figure 8). SEM microscopy support this result, suggesting that mainly two or three SLN are involved in the complex formation with RNA (Figure 4B). As shown in gel shift experiments, depending on the cationic SLN-RNA w/w ratio, complexes were produced in a selfassembling manner. Thus, at an SLN-RNA w/w ratio of 200:1 the negative charges on RNA, as also confirmed by ζ-potential measurements, are neutralized and the charge complex is positive. This is relevant for the bioelectrochemistry and the biodistribution of RNA-nanoparticles complexes, increasing their adsorption efficiency in the negatively charged cellular membranes. Moreover, nanoparticles are able to protect bep3 by RNAase T1 enzymatic degradation even at high concentration and by RNAse A in concentration consistent with a cellular environment, strengthening the idea of a high affinity for RNA molecules. This might permit major RNA stability inside the cellular environments that results in a required condition for nanobiotechnology applications. To utilize SLN for eventual medical applications, we need to understand if they have a potential toxic effect. To address this question, we decided to utilize the sea urchin as a model system. This organism is very sensitive to different chemical or environmental agents (31, 32). Microinjection of cationic nanoparticles into the Paracentrotus liVidus eggs permits normal embryonic development as observed by their high viability, regular cell cycle, and correct morphogenesis, indicating their high biocompatibility within the cellular environment. Thus, cationic SLN are composed by physiologically well tolerated ingredients, indicating that they may be suitable for pharmacological applications. Moreover, we took advantage of the

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characteristic movement of bep3 RNA along the cytoskeleton to test the potential delivering ability of SLN. The observed correct biodistribution of the RNA suggests that the +/- charge ratio and the ζ potential of the complexes allows an efficient transfer of bep3 into the cell that maintains its functionality. All these data, together with the ability of SLN to diffuse through the cellular membrane and its potential controlled release of a bioactive molecule, such as bep3 RNA, suggest that they may have specific characteristics for being a good tool for gene delivery in human therapy.

ACKNOWLEDGMENT We thank Mr. Alessandro Pensato for technical assistance.

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