Quaternary Ammonium Groups Exposed at the Surface of Silica

Article pubs.acs.org/JPCB

Quaternary Ammonium Groups Exposed at the Surface of Silica Nanoparticles Suitable for DNA Complexation in the Presence of Cationic Lipids Nora Reinhardt,†,‡,∥ Laurent Adumeau,†,∥ Olivier Lambert,§ Serge Ravaine,‡ and Stéphane Mornet*,† †

CNRS, ICMCB, UPR 9048, Université de Bordeaux, 87 avenue du Dr. A. Schweitzer, F-33600 Pessac, France CNRS, CRPP, UPR 8641, Université de Bordeaux, F-33600, Pessac, France § CNRS, CBMN, UMR 5248, Université de Bordeaux, F-33402 Talence, France ‡

S Supporting Information *

ABSTRACT: The production of silica nanoparticles (NPs) exposing quaternary ammonium groups (NPQ+) has been achieved using an optimized chemical surface functionalization protocol. The procedures of surface modification and quaternization of amino groups were validated by diffuse reflectance infrared Fourier transform (DRIFT) and 1H NMR spectroscopies. Compared to nonquaternized aminated NP, the colloidal stability of NPQ+ was improved for various pH and salt conditions as assessed by ζ potential and light scattering measurements. In the context of their use for nucleic acid delivery, DNA efficiently bound to NPQ+ analyzed by cosedimentation assays for a large pH range and various NaCl concentrations and exhibited a better efficacy at basic pH than nonquaternized NP. The study of NPQ+/DNA/cationic lipids ternary complexes was carried out with 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and analyzed by cryo-electron microscopy (cryo-EM). Cryo-EM images showed ternary assemblies where condensed DNA strands are sandwiched between the NPQ+ surface and the cationic lipid bilayer. Because of an unusual electrostatic colloidal stability of NPQ+ and a high propensity to bind DNA molecules particularly at high salt concentrations, a novel type of ternary assembly has been formed that might impact the delivery properties of these complexes including their stability in biological environment. sandwiched between cationic lipid bilayers.6−8 This strategy allowed to track this gene vector at various scales up to subcellular level.5,9,10 In terms of transfection efficiency, aminemodified silica NPs has been tested11 and it has been shown that the adsorption of DNA molecules on the nanoparticles prevents it from nucleases cleavage in vitro and in cellular environment.12,13 Using cationic aminated silica NPs, it has been also reported that at high NP/plasmid DNA ratios (>10), transfection efficiency was enhanced.14 Other examples involving gold NPs bearing positively charged moieties such as spermine or spermidine15 and gold nanorods coated by a cationic lipid bilayer16 complexed with DNA and RNA have showed transfection abilities. A milestone in the design of silica nanoparticles used in gene transfection is their surface modification since their nude surface is negatively charged. The issue raised here is that nanoparticles should present sufficiently strong interactions to condense nucleic acid molecules to their surface. For example, in the context of Lx labeling these interactions have to be

1. INTRODUCTION Over the past decade, nonviral transfection methods implementing natural or synthetic positively charged molecules have been extensively developed to collapse extended DNA chains into compact complexes.1 These molecules are often multivalent cations such as cationic polymers, polyamines, peptides, or cationic amphiphilic molecules used to neutralize the overall charges of the phosphate backbone at the origin of the DNA condensation.2 The use of cationic inorganic nanoparticles (NPs) has emerged more recently in gene therapy, in particular, as gene vector labeling tools as well as new way to improve the transfection process. The precise mechanisms of gene transfer with cationic vectors remain ill-defined.3 By providing access to complementary imaging modalities with high sensitivity or high spatial resolution,4 NPs constitute real tools to help in a better understanding of the transfection processes. Among them, silica based NPs is a good candidate for the development of multimodal tool since the silica matrix can host a wide variety of probes such as fluorescent dyes and semiconductors or magnetic NPs. For example, cationic silica NPs have been incorporated in lipoplexes (Lx) structures5 consisting of highly ordered multilamellar assembly with rod shaped DNA, © 2015 American Chemical Society

Received: February 24, 2015 Revised: May 6, 2015 Published: May 7, 2015 6401

DOI: 10.1021/acs.jpcb.5b01834 J. Phys. Chem. B 2015, 119, 6401−6411

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The Journal of Physical Chemistry B

Synthesis of Silica Nanoparticles. The 50 nm diameter silica nanoparticles were produced in two steps by a seed growth method described as follow. First, seeds are prepared by rapidly adding under continuous stirring a volume of 6 mL of TEOS dissolved in 270 mL of EtOH into 302 mL of alcoholic solution of deionized H2O and NH4OH, at a volume ratio of 89.5/0.9/9.6 for absolute EtOH, H 2 O, and NH 4 OH, respectively. The mixture was stirred for 2 h at room temperature. Silica nanoparticles with diameters of 11.4 ± 1 nm were obtained. A volume of 200 mL of the freshly prepared seeds dispersion (1.4 g/L) were introduced to 1.02 L of reaction medium consisting of 71.3% v/v EtOH, 27.3% v/v H2O and 1.3% v/v NH4OH. Then, 42.8 mL TEOS were added under continuous stirring at room temperature during 4 h. Two hours after the end of the addition, NH4OH and EtOH were removed from the medium by evaporation at 40 °C under reduced pressure. The size of the grown nanoparticles was determined by TEM, and the concentration of silica NPs/L inductively coupled plasma−optical emission spectroscopy (ICP−OES). Silica nanoparticles with diameters of 50 ± 3 nm were obtained. Surface Modification of Colloidal Silica with Aminosilanes Coupling Agents (SiO2-EDPS). Surface modification of silica nanoparticles were carried out by adding in the medium an excess of 10 equiv of EDPS, sufficient to provide 2−3 monolayer coatings of the silica nanoparticles. Starting from the developed surface area of the nanoparticles and given that the area on the nanoparticle surface covered by the organosilane coupling agent is assumed to be nominally 55 Å2 per molecule,21 it is possible to calculate the amount of organosilane needed for a given nanoparticle size. After it was left to react overnight, 100 mL of glycerol were added and the ammonia and ethanol were evaporated at low boil under a moderate vacuum. In order to promote the condensation of the polysiloxane film to the silica surface, the reaction was followed by a thermal treatment in vacuum at 100−110 °C. The modified particles were washed four times with absolute ethanol by centrifugation at 13000 g for 25 min before dispersion in final volume of 100 mL of absolute ethanol. Methylation of Primary and Secondary Amines Present on SiO2-EDPS Nanoparticles (SiO2-EDPS-Me/ NPQ+). To a volume of 10 mL of SiO2-EDPS NPs of 26.4 m2 surface area dispersed in absolute EtOH, 220 μL of Et3N were added under continuous stirring and subsequent 410 μL of iodomethane were added dropwise. The reaction mixture was stirred for 12 h at room temperature and for 30 min at 70 °C in a water bath. The particles were then washed twice with absolute EtOH, once with EtOH/H2O (50% v/v) and subsequent two times with H2O (18.2 MΩ) by centrifugation during 25 min at 13000g. Iodide counterions remaining in the solution were precipitated by the addition of AgNO3. After recovery of the modified particles two additional washing steps were done in H2O (18.2 MΩ) by centrifuging 25 min at 13000g. DRIFT Spectroscopy. A volume of the nanoparticle sol was dried at 80 °C under vacuum to obtain the sample under a powder form. Then, samples to be analyzed were prepared by spreading crushed powders (3 wt %) in anhydrous (spectroscopy grade) KBr on a conical support. Spectra were recorded using a Bruker IFS Equinox 55 FTIR spectrometer (signal averaging 32 scans at a resolution of 4 cm−1) equipped by a selector Graseby Specac diffuse reflection cell (Eurolabo, France).

strong enough so as to avoid their exchange by cationic lipids during their elaboration while satisfying DNA release reversibility conditions that must occur in the cytosol. The most commonly used functional surface agents as DNA condensation agents are implementing amines or polyamines groups on the particle surfaces. Quaternary ammonium groups are also well-known to possess a high attraction for the adsorption of phosphate groups containing compounds such as DNA.17,18 In order to place quaternary ammonium functional groups on silica, multiple efforts have been implemented to graft quaternary ammonium bearing silane coupling agent Ntrimethoxysilylpropyl-N,N,N-trimethylammonium chloride on silica nanoparticles. However, such functionalized silica particles possess often isoelectric points in a pH range from pH 6.7 to pH 8.4 attesting to the presence of negatively charged silanolate surface sites which decrease the electrostatic surface potential and lead to a lack of colloidal stability.19,20 In the present study, a three-step procedure leading to NP/ DNA/lipid complexes is described. We used surface-modified silica nanoparticles of 50 nm sized bearing primary/secondary or quaternary amino groups as cationic surface site for DNA binding. First, we exposed the approach of chemical surface modification of silica nanoparticles leading to surfaces-bearing quaternary ammonium sites (called also thereafter “quaternized” nanoparticles, NPQ+), which enables to drastically enhance the adhesion with DNA macromolecules. All the surface chemistry steps were validated by diffuse reflectance infrared Fourier transform (DRIFT) and proton nuclear magnetic resonance spectroscopy (1H NMR) spectroscopies. Then the physicochemical properties of these cationic colloidal dispersions were thoroughly characterized, using ζ potential and light scattering measurements, to explain the behavior differences between both types of ammonium group-bearing silica surfaces in terms of DNA binding. In a second part, the interactions between cationic surfaces possessing quaternary or primary/secondary ammoniums and DNA macromolecules were characterized using cosedimentation assays. Third, cationic liposomes were interacted with NPQ+/DNA aggregates leading to NPQ+/DNA/Lipid complexes with multilamellar lipoplexes. The results showed morphological changes of the Lx formed on the colloidal surfaces attributed to the significant differences of DNA conformations displayed for high and low ionic strength conditions. These new complexes could display interesting original transfection properties offering new endocytosis and DNA release pathways.

2. EXPERIMENTAL METHODS Chemicals, DNA, and Cationic Liposomes. N-[3(Trimethoxysilyl)propyl]ethylenediamine (EDPS, 97% w/v), tetraethylorthosilicate (≥99.0%, TEOS), glycerol (≥99.5%), deoxyribonucleic acid (DNA) from herring sperm, silver nitrate (≥99.0%), sodium chloride (≥99.5%), uranyl acetate (≥98.0%), iodomethane (99%), ethanol (EtOH, ≥99.8%), trimethylamine (Et3N, ≥99.0%), and anhydrous acetonitrile (99.8%) were purchased from Sigma-Aldrich (Saint-QuentinFallavier, France). Ammonium hydroxide (NH4OH, 28−30% w/v) was furnished by Atlantic labo Inc. (Bruges, France). 1,2Dioleoyl-3-trimethylammonium propane (DOTAP) was obtained from Avanti Polar Lipids. In all the experiments, water was previously deionized (18 MΩ). Except for the DNA from herring sperm, which has been classically purified of proteins according to the procedure described in the Supporting Information, all reagents were used as received. 6402


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solutions were buffered to fix the pH and adjusted to a desired salt concentration. Samples adjusted to pH 3.5 and pH 6 were buffered in MES buffer (10 mM), samples at pH 7.4 in HEPES buffer (10 mM) and samples at pH 8.6 and pH 9 in borate buffer (10 mM). A control sample without the presence of nanoparticles was prepared. After 15 min of incubation time the samples were centrifuged at 21000g for 15 min. DNA concentration in the supernatant was determined by a spectrophotometric measurement at 260 nm with a NanoDrop 1000 spectrophotometer. The percentage values of cosedimented DNA derive from those of the DNA concentration measured in the control sample. NPQ+/DNA/DOTAP SUVs Complexation. A volume of 10 μL of an aqueous dispersion of SiO2-EDPS-Me (called also NPQ+) (1.72 × 1017 NPs/L) was added under careful agitation to 90 μL aqueous solution containing 5 μg DNA and 10 μL MES buffer (100 mM, pH 6.5, with 150 mM NaCl or without NaCl) in order to obtain NP/DNA complexes at 0.53 (nm2/ bp) NP/DNA ratio. A volume of 1.89 μL of SUVs dispersion (DOTAP, 10 mg/mL) was added to 30 μL of the NP/DNA complex dispersion which corresponds to a SLB/SNP surface ratio of 6. Conventional TEM and cryo-EM. Particles were deposited on a carbon-film covered copper grid. A series of TEM micrographs was performed with a Philips CM120 microscope operating at 120 kV and captured with the Ultra scan USC1000 2k × 2k camera. For cryo-EM, 5 μL of sample were deposited onto a holey carbon coated copper grid. The excess was blotted with a filter paper. Samples were frozen into liquid ethane and the grids were mounted onto a Gatan 626 cryoholder, transferred into the microscope, and maintained at a temperature of around −175 °C. Sample observations were performed with a FEI Tecnai F20 transmission electron microscope, operating at 200 kV. Low-dose images were recorded at a nominal magnification of 50,000× with a 2k × 2k USC1000 slow-scan CCD camera (Gatan, CA). Measurements of NP sizes and supported lipid bilayer thicknesses were performed with the Digital Micrograph software from Gatan.

1

H NMR Spectroscopy. First, 1.5 mL of aqueous nanoparticle sol was centrifuged for 10 min at 13000g in 2 mL Eppendorf cups. The supernatant was removed and nanoparticles were redispersed in 1 mL of D2O. A second step of washing with D2O was applied by centrifugation and nanoparticles were redispersed in 500 μL of D2O. All spectra were recorded in a Bruker NMR spectroscope at 9.4 T (400 MHz). A total of 128 scans were implemented to record one NMR spectrum. Conductometry. Conductivity measurements were carried out using a Bioblock Scientific conductivity meter 93008 to titrate halide ions. A titration solution of AgNO3 (0.1 M) was freshly prepared. Immediately after quaternization, two washing steps were accomplished in ultrapure water. A volume of 2 mL of a dispersion containing surface modified nanoparticles was withdrawn. The conductivity of the sol was measured after each addition of 10 ± 0.1 μL of the AgNO3 solution. Dynamic Light Scattering (DLS). Around 1 mL of a diluted sol of 7 × 1015 NPs/L was introduced to the VASCO particle size analyzer from Cordouan Technologies (Pessac, France). The laser intensity of the 658 nm laser diode was adjusted and the sample cooled to a temperature of 20 °C. The refractive index and viscosity of the solvent as well as the refractive index of the particles were entered into the software NanoQ, which pilots the measurements. A real time signal was measured; acquisitions were implemented every 30 s if the noise of the signal did not exceed 1.06%. 80 measurements were accomplished to determine size distribution and the polydispersity index of the nanoparticles by the cumulant method. ζ Potential Measurements. Nanoparticle dispersion was diluted to a concentration of about 3.4 × 1016 NPs/L. The pH of the solution was adjusted by the addition of HCl (0.01 M) or NaOH (0.01 M). At the desired pH a volume of 5 mL of the sample was collected. After pH equilibration for 12 h, pH of the samples were measured once again and the ζ potential values were measured using the Malvern Zetasizer 3000 HS setup (Malvern Instruments) equipped with a He−Ne laser (50 mW, 532 nm). Each measurement was performed for 20 s, the dielectric constant of solvent (water) was set to 80.4 and the Smoluchowsky constant f(ka) was 1.5. Formation of Small Unilamellar Lipid Vesicles (SUVs). First, 400 μL of a DOTAP lipid stock solution in chloroform (50 mg/mL) was taken with a glass syringe and added into a 5 mL round-bottom glass flask. Chloroform was evaporated at room temperature to form a thin lipid film on the wall of the glass flask. In order to remove traces of chloroform, twice the lipid film was dissolved in 1 mL of diethyl ether and formed again by evaporation of the solvent. Then, the lipid film was dried in a desiccator under vacuum for 20 min at room temperature and hydrated by addition of 2 mL of ultrapure water (18.2 MΩ) under 1 min vortexing. The emulsion was frozen in liquid nitrogen at −200 °C and thawed out in a 37 °C water bath. The freezing thawing cycle was repeated four times. The sample was placed in an ice bath. Sonication pulses of two seconds with 20% amplitude were applied for 30 min using a tip sonicator vibra cell 75042 from Bioblock Scientific to generate SUVs. Titanium fragments detached from the sonication tip were removed from dispersion by centrifugation at 13000g. Co-Sedimentation Assays. First, 60 μL of a diluted dispersion of surface-modified nanoparticles was added to 40 μL of a solution containing 5 μg of herring sperm DNA. Both

3. RESULTS AND DISCUSSION 3.1. Chemical Surface Modification and Quaternization of Aminated Silica NPs Surface. Silica nanoparticles of 50 nm diameter were produced for this study. The choice of this nanoparticle size is justified by practical raisons, i.e., it is well adapted for cosedimentation assays for which NPs can be easily isolated from free DNA molecules by centrifugation and this is also an ideal size for subsequent cryo-EM observations. Their synthesis consists of a modified Stö ber method implementing a seed growth approach used in order to produce upon request nanoparticles of same size with a good reproducibility. Chemical surface modification of silica nanoparticles by silanization with EDPS was performed according to a method previously described.22 The alkylation reaction of amine groups with iodomethane can take place in different solvents and can be accomplished with different bases or alkaline buffers such as NEt3 and K2CO3 respectively.23 In colloid chemical surface modification, optimal conditions for carrying out the organic reaction have to be adapted with those allowing the dispersion of the nanoparticles during each synthesis stage. The methylation of silica nanoparticles bearing amine groups has already been effected in the literature using acetonitrile as a solvent and K2CO3 as a base,24 but we 6403


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observed that EDPS-modified nanoparticles could not be dispersed in acetonitrile and the methylation performed with K2CO3 systematically led to the destabilization of the dispersions. In this study, quaternization reactions were performed in EtOH using NEt3 as base which gave better results. At the end of methylation reaction, quaternized nanoparticles spontaneously dispersed in water without the need of any acid usually used to peptize the particles by protonation of the primary and secondary surface amino groups. This behavior was expected because the permanent presence of the pH independent positive charges of quaternary ammonium groups permits the immediate dispersion and stabilization of the modified nanoparticles in ultrapure water. Both nanoparticle surfaces before and after amine quaternization were characterized by DRIFT and 1H NMR spectroscopies. Figure 1 shows the infrared spectra of EDPS modified

H NMR study performed on colloidal dispersions after each step of chemical surface modification confirms also the success of the alkylation. The 1H NMR spectrum of EDPS-modified silica (Figure S2) shows the characteristic peaks of the 3-(2aminoethylamino)propyl group which are not present on the nude colloidal silica (Figure S1). The spectra of the same EDPS-modified particles after alkylation with iodomethane (Figure S3, and COSY 2D spectrum in Figure S4) confirm the successful quaternization of primary and secondary amine groups with in particular, additional characteristic peaks coming from the 3-(2-trimethylammonium-ethyldimethylammonium)propyl moieties (Figure S3). Integration of peaks gives more additional interesting information about the functionalization. In the 1H NMR spectrum shown in Figure S3, the intensity of the two signals attributed to H1 and H4 atoms next to quaternary ammonium groups is about nine times higher than the intensity of the signals of H1 and H4 atoms next to tertiary amine groups, which indicates that the quaternization of amine groups is nearly complete. Integration of the four singulets shows an integral of 12.73 and 7.33 for the quaternary proton peaks in contrast to integrals of 1.12 and 1.0, which is a repartition of 92% of quaternized ammonium groups to 8% of tertiary amine groups at position 1 and 88% of quaternized ammonium groups to 12% of tertiary amine groups at position 4. The NH peak of H1 and H4 expected at 2.5 ppm is not observed, which is also explained by the fast exchange of NH protons with deuterium of the solvent D2O. Therefore, it is not possible to determine the yield of alkylation of the propylethylenediamine grafts, but the results show that about 90% of alkylated amine groups are in a quaternized form. Thus, DRIFT and NMR spectroscopy studies have revealed the presence of quaternary ammonium groups on methylated EDPS-modified silica nanoparticles. Conductometric titration of particle surrounding iodide counterions was performed to determine the surface grafting density of quaternized amino groups (Figure S5). Supposing that each quaternary ammonium group is accompanied by one iodide ion serving as a counterion, the number of quaternary ammonium groups can be indirectly estimated by a conductometric dosage of iodide ions present in the dispersion. Addition of AgNO3 solution (0.1 M) leads to the precipitation of AgI due to the very weak solubility constant of this solid in water (KS = 8.52 × 10−17 at 298 K). When the total amount of iodide has reacted, no more precipitation of AgI takes place and, by that time, the added Ag+ ions start to contribute to the increase in the conductivity of the dispersion. The value at the turning point corresponds to the volume and so the number of moles in I− counterions for an amount of silica nanoparticles with a total surface area developed known. In the same way the amount of EDPS grafts on EDPS-modified silica nanoparticles peptized with hydrochloric acid has been dosed by the precipitation of AgCl (KS = 1.77 × 10−10 at 298 K). After synthesis, all samples were washed in ethanol before immersing in ultrapure water. The peptization of nanoparticles occurs spontaneously without any addition of acid. The measured pH value is about of 4.7 due to the HI formation coming from ion exchange between remaining acidic silanol sites27 of polysiloxane film in interaction with a part of quaternary ammoniums groups. The total amount of iodide or chloride counterions estimated in this medium respectively corresponds to the number of quaternary or primary/secondary ammoniums present on the nanoparticle surface. This method permitted to find 6.8 μmol

Figure 1. (a) DRIFT spectra of aminated SiO2-EDPS and methylated SiO2-EDPS nanoparticles (SiO2-EDPS-Me). (b and c) Zooms to regions with vibration bands characteristic for the organic grafts on particle surface.

silica nanoparticles (SiO 2-EDPS) before and after the methylation with iodomethane (SiO2-EDPS-Me). In the range from 1500 to 1390 cm−1 it can be distinguished four vibration bands. The bands at 1395 and 1418 cm−1 are attributed to C−N deformation vibrations; the band at 1395 cm−1 shifts to higher wavelengths in the presence of quaternary ammonium ions. In the same manner the band of C−H deformation vibrations shifts from a value of 1452 cm−1 if the carbon chain is attached to a primary or a secondary amine group, to a value of 1479 cm−1 in the presence of quaternary ammonium groups.25,26 Bands in the range of 2900−2980 cm−1 are attributed to C−H stretching vibrations of the methyl groups attached to a nitrogen atom and aliphatic hydrocarbons from aminosilane film. 6404


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the thermal treatment, the condensation of polysiloxane film all around the particle surface is completed by minimization of the intramolecular interactions between silanol and the amino groups.30−32 Figure 2b shows scattering measurements performed at 800 nm, wavelength where aminated and quaternized nanoparticle dispersions do not absorb. So, an increase of the optical density (OD equal to −log T, and so a decrease of the transmittance) is related to a higher light scattering reflecting the presence of more particles agglomerated while an OD of zero corresponding thus to a transmittance of 100%, is observed for a transparent stable colloidal dispersion. It is observed that the presence of such protonable amino groups in the neutral pH conditions ensures the electrostatic colloidal stability up to a pH of about 7−7.5 (Figure 2b). The quaternization of primary and secondary amine groups of EDPS-modified surfaces leads to pH independent surface potential with higher value (of about 50 mV) up to the beginning of the alkaline zone. A shift of the IEP from 9 to 10.5 is also observed traducing differences of colloidal stability behaviors between both dispersions. In contrast to EDPSmodified nanoparticles, which coagulate in the pH range from 7.5 to 10.5, the coagulation zone is shifted to a pH range from about 9 to 11.5 for quaternized particles (Figure 2b). This effect is explained by the pH independence of quaternary ammonium groups in comparison with primary and secondary ammonium groups, which are sensitive to the deprotonation in the presence of a base. The destabilization of such quaternary ammonium-modified silica nanoparticles is explained by the dissociation of silica in alkaline medium. Because of the dissociation constant of silica of 10.5, an increase of the pH causes the apparition of more and more negatively charged surface silanolate groups. This dissolution of silica in basic medium explains the presence of an isoelectric point for quaternized silica nanoparticles and the possibility to destabilize these dispersions probably through electrostatic intramolecular interactions between quaternary ammoniums and silanolate groups. Another even more remarkable result lies in the weak effect of ionic strength on colloidal stability of the quaternized samples (Figure 3a). Indeed, while EDPS-modified silica dispersions display a sensitive charge screening behavior to colloidal destabilization for weak NaCl concentrations up to 10 mM at pH 6.9, quaternized silica dispersions are very stable even at high salt concentrations up to 300 mM NaCl for weeks at the same pH. ζ potential profile vs pH performed at 150 mM NaCl is not affected by the screening of the salts (Figure 3b). Moreover it is noteworthy that cationic charges of quaternized silica NPs are independent from environmental conditions in acidic media until pH 7 (Figure 2a) with a quite high ζ value of 50 mV. Structural and physicochemical considerations have to be exposed here in order to give explanations of these behaviors. First, the physicochemical nature of these two cations is very different, for example, as evidenced by the location of the positive partial charge mainly brought by protons for primary ammonium and by the nitrogen for quaternary ammoniums. In terms of solvation features, both polar and nonpolar interactions contribute to hydration, i.e., the immediate vicinity of water molecules around the ions, of primary and quaternary ammonium.33 For primary ammonium, the water structure around amines is dictated by the hydrogen bond formation of the polar group with water. In the case of quaternized

of chloride ions per m2 corresponding to 3.4 μmol of EDPS per m2 of nanoparticle surface area, or 49 Å2/molecule, assuming that both amino groups of EDPS graft are protonated at this pH. This value is close to the value of 55 Å2/molecule given in literature.31 In the case of quaternized nanoparticles a value of 4.3 μmol of iodide counterions per m2 has been measured corresponding to a charge density of 2.6 charge/nm2. A quite close value of cationic charge density of 4.5 μmol/m2 (i.e., 2.7 charge/nm2) has been obtained by weighing of the AgI precipitated. These results give a quaternization yield in the range from 63 to 66% for completely alkylated ammonium. This result is explained by the fact that all amino groups inside the polysiloxane film are not accessible because of steric hindrances. 3.2. Physico-Chemical Features of Quaternized Silica Colloidal Dispersions. ζ potential measurements of bare and aminated silica nanoparticles before and after methylation have been performed in order to provide and compare the physicochemical characteristics of both surfaces. The ζ potential curve (Figure 2a) of silica nanoparticles displays an

Figure 2. (a) Comparison of the ζ potential versus pH of silica nanoparticles before and after EDPS surface treatment and after methylation (SiO2-EDPS-Me). The isoelectric point shifts from pH 9 to pH 10.5. (b) Colloidal stability of aminated silica sols vs NaCl concentration. The dotted lines are just guides for the eye.

isoelectric point (IEP) at pH 2.6, in agreement with previous reports (IEPs of silica are in the pH range of 2−3).28 After silanization of the silica surface by EDPS, the so-modified nanoparticles display a quite high ζ potential value of about 33 mV at pH 7.4 with an IEP at pH 9 that corresponds to a value close to the pKa of the primary aminopropyl group reflecting an optimal condensation of polysiloxane film around particles with a minimal number of remaining silanolate groups.29 Because of 6405


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Figure 3. (a) Colloidal stability of primary/secondary (SiO2-EDPS) and quaternary aminated (SiO2-EDPS-Me) silica nanoparticle dispersions vs NaCl concentration for different pH values. (b) Comparison of ζ potential versus pH of SiO2-EDPS-Me nanoparticles in presence and absence of NaCl. The lines are just guides for the eye.

i.e., F− > Cl− > Br− > I− while for tetraalkylated cations this series, however, reverses.35 In other terms, moving down the periodic table, pairing of primary ammonium with halide ions becomes gradually weaker while the pairing is reversed for quaternary ones. More generally, this result is in accord with the empirical rule stating that large cations tend to pair with large anions.36 This property is interesting to exploit in the frame of the adsorption of DNA polyanions on the quaternized NP surface. These considerations would also explain the trend of quaternary ammoniums to be far less sensitive to the screening by Cl− counterions either by varying the pH in acidic medium, i.e., the HCl concentration, that by varying the NaCl concentration (Figures 2a and 3). 3.3. Characterizations of NP/DNA Interactions and DNA Binding Capacity of NPQ+. DNA binding capacity of nanoparticles was assessed by cosedimentation assays using a Nanodrop spectrophotometer. The influence of the pH and NaCl concentration on the ability of modified nanoparticles to

ammoniums, the hydration is strongly influenced by the interactions of alkyl groups with water.34 In their study Rao and Singh have reported from energy free perturbation calculations that the total free energy decrease from −100.2 to −117.8 kcal/ mol when one goes from Me4N+ to MeNH3+. This decrease in free energy is due to both favorable entropic and heats of hydration contributions cause by the tightly bound water structure and the enhancement of solute−solvent interaction, respectively. In other words compared with primary ammonium, quaternary form behaves as a “hard” core, where water molecules penetrate less and alkyl moieties provides further sterical constraints to the water spatial distribution, resulting in a lower number of water molecules. A direct consequence of that lies in the pairing of the chloride counterion which is drastically different between ammonium and alkylated ammonium cations in water. It has been recently reported from molecular dynamics simulations that (primary) ammonium cations follow the Hofmeister series, 6406


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Figure 4. (a) Co-sedimentation assays showing the effect of the pH on the capacity of NPQ+ (SiO2-EDPS-Me) to complex DNA strands in 150 mM NaCl, 10 mM buffers: pH 3.5 and pH 6 (MES); pH 7.4 (HEPES); pH 8.6 and pH 9 (borate). TEM micrographs show nanoparticle DNA complexes formed at different nanoparticle/DNA ratios at pH 7.4 and 150 mM NaCl. The specimens were stained with uranyl acetate solution. Ratio A refers to NP/DNA complexes assembled at 0.3 nm2/bp (black arrow A in graph). White arrows in the inset show free DNA strands in the supernatant recognizable next to the SiO2-EDPS-Me/DNA complexes. Ratio B refers to NP/DNA complexes assembled at 2.5 nm2/bp (black arrow B in graph) (scale bar =1 μm, scale bar of inserts =200 nm). (b) Co-sedimentation of DNA by modified nanoparticles in relation to the salt concentration (in 10 mM HEPES buffer at pH 7.4). Inset showing the region from 0.1 to 1 nm2/bp. (c) Comparison of the amount of EDPS-modified or quaternized nanoparticles needed to complex 50% of DNA during a cosedimentation assay (r50) for various pH values. (d) Co-sedimentation of DNA by quaternized EDPS-modified silica nanoparticles in relation to an increasing of dextran sulfate (Mw > 500000) concentration at pH 7.4 in the presence of 150 mM NaCl. In all graphs, the dotted lines are just guides for the eye.

natant after the centrifugation of nanoparticle/DNA complexes at different pH, it was observed that a diminution of the interactions between DNA and NPQ+ in alkaline media (Figure 4a) took place. As expected, the increase in pH leads to a diminution of the amount of DNA which can be bound per surface unit of the nanoparticle surface area. As a consequence the surface area which is needed to bind 100% of the disposed DNA increases. At pH 3.5 nanoparticle surface area of 0.7 nm2 is needed to complex one DNA base pair, which corresponds to an ideal binding of DNA in view of the fact that one base pair requires 0.68 nm2. Whereas at pH 6.5 the cosedimentation of

complex DNA has been investigated (Figures 4a and 4b). With increasing pH, the ζ potential of primary/secondary aminated (SiO2-EDPS) as well as of quaternized nanoparticles decreases, which should have a direct influence on their capacity to interact with DNA. The cosedimentation assays were implemented at an electrolyte concentration comparable to the concentration of salts in the cytosol. This has the advantage that the salt screens are a part of the charges of the deoxyribose phosphate backbone of the DNA, rendering the strands less stiff and more flexible.37,38 In measuring the DNA concentrations remaining in the super6407


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Figure 5. Cryo-EM (a, b, d, e) and uranyl acetate stained TEM (c, f) images of NPQ+/DNA/cationic lipids agglomerations (ratio NP/DNA = 0.53 nm2/bp, ratio NP surface area/lipid surface = 1/6) formed at pH 6.5 in 10 mM MES buffer at 150 mM NaCl (a, b, c) and without NaCl (d, e, f). Different assemblies occur: The gray arrows show region where dots which could correspond to DNA cylinders are located between the cationic nanoparticle surface and the cationic lipid bilayer, lamellar Lx structures are indicated by black arrows (circular, concentric structures) and white arrows (planar and angular structures). In picture b, the inset shows a higher magnification (scale bar 50 nm) where the gray arrows show regions which correspond to DNA cylinders located between the cationic nanoparticle surface and the cationic lipid bilayer. This interspace layer is around 2 nm thick that fits quite with double-stranded DNA diameter.The drawings illustrate the different scenarios between DOTAP SUVs and NP/DNA complexes: (a) Wrapping of DNA to circular Lx while coating the NP; (b) wrapping of stiff DNA molecules to planar Lx which embeds a part of the NP surface. All Cryo-EM pictures are at the same magnification, scale bar = 50 nm.

100% of DNA is reached at a ratio of 0.9 nm2 per base pair, which is near to the ideal case observed at pH 3.5, a diminution of the DNA-binding-capacity of NPQ+ at the physiological pH 7.4 is observed, where 1.3 nm2 is needed to complex one DNA base pair. The further augmentation of the pH has an increasing influence of the DNA bindingcapacity of the nanoparticles due to the more rapid decrease of their ζ potential in alkaline medium. Figure 4c shows the ratio r50 corresponding to the amount of nanoparticle surface needed to complex 50% of DNA. It is observed that at alkaline medium above pH 8, more aminated nanoparticles than quaternized nanoparticles are needed to bind 50% of DNA present in the sample. This trend follows ζ potential variations with the pH (Figure 2a) of both surface-modified silica nanoparticles. The quaternary ammonium groups on the particle surface enable electrostatic interactions that take place even under alkaline conditions. Transmission electron microscopy was used to examine the size variation of nanoparticle/DNA complexes for a given surface area per base pair (Figure 4a). For the ratio A, corresponding to 0.3 nm2/bp, the remaining DNA strands are clearly distinguished in the supernatant next to the small nanoparticles aggregates. Then, the size of the nanoparticle/

DNA complexes increases with the increase of the ratio of nanoparticles to DNA (Figure 4a) up to the ratio used for the complete complexation of DNA. This result is in accordance with previous observations in the literature5,11,13 with aminated nanoparticles complexed with plasmidic DNA to enhance the DNA transfection efficiency.11,13 Considering the fact that NPQ+ are highly stable for high salt concentration (Figure 3) and that the flexibility and conformation of a DNA strand can be influenced39 for high ionic strengths, cosedimentation experiments were implemented at pH 7.4 for different NaCl concentrations (Figure 4b). The cosedimentation experiments showed that with increasing salt concentration fewer nanoparticles are needed to complex 100% of the present DNA. For these conditions of salt, the level of intramolecular electrostatic repulsions between two strands is reduced by shielding, resulting in a decrease in the persistence length and thus the stiffness which impacts directly the DNA complexation with nanoparticles.37,38 The complex stability was investigated by a competitive exchange of DNA adsorbed to the nanoparticles and dextran sulfate. It is known that dextran sulfate can be used to extract histone depleted chromosome DNA.40 We measured the 6408


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inset, and S7), value which quite fits with the diameter of a double-stranded DNA helix. In addition the formation of SLB of DOTAP over quaternized nanoparticle surface can only be explained to the presence of negatively charged DNA molecules which allies the two positively charged compounds. Indeed, given that the ζ potential value of DOTAP SUVs at pH 6.5 and 150 mM NaCl was measured at 60 mV and those of quaternized amino-nanoparticles are 40 or 50 mV according to the ionic strength (Figure 3b), supporting bilayers cannot form directly on these nanoparticle surfaces due to electrostatic repulsion forces. The presence on the NP surface of cationic headgroups similar to those of the lipid is favorable to fix strongly the DNA molecules on their surface and prevent the partial or complete detachment of DNA molecules from the surface during the complexation. It was also observed the formation of multilamellar concentric structures as indicated by black arrows around the particles which are related to lipoplextype (Lx) structures (Figures 5, parts a, b, c). In control experiments performed without DNA macromolecules under the same conditions, it is clearly evidenced that both cationic colloidal systems do not interact (Figure S8, parts a and b). In this case no “suspended” SLB and obviously no Lx formation onto quaternized nanoparticle surfaces were observed, which tends to demonstrate that DNA is necessary to form these both structures. In the absence of salt, in addition to such “suspended” supported lipid bilayers which appear less frequently (Figure 5, parts d, e, and f, indicated by black arrows), it could be discerned predominating multilamellar LCα lipoplex phases adsorbed on quaternized nanoparticles or sometimes surrounding them partially. These phases are commonly observed for such mole fractions of cationic lipids (here ϕ > 0.99) and lipidto-DNA charge ratios (ρ = 1.59) used for their formation.48 These Lx previously observed49,50 exhibit angular and planar morphologies indicated by white arrows in Figure 5, parts b, d, and f. The planar Lx layers commonly observed for DOTAP lipid which possess a small headgroup area, is due to the difference of stiffness of DNA molecules. In this case, DNA molecules are in elongated coiled state with linear less flexible double strands and longer persistence lengths. These molecules partly interact with the NP surfaces which explain the partial Lx coating on cationic nanoparticle surfaces. Control experiments performed for these conditions without DNA macromolecules clearly showed that both cationic colloidal systems do not interact (Figure S8, parts c and d). As for the previous case in the presence of NaCl, neither the SLB and nor the Lx supramolecular assemblies onto quaternized nanoparticle surfaces were observed which demonstrates again that DNA is necessary to form these both structures. As a result it has been demonstrated that thanks to their high colloidal stability in the presence of salts and high DNA binding capacity, NPQ+ enable to study the tripartite complexation of cationic colloidal surfaces, DNA molecules and cationic lipids. Two distinct morphologies have been thus depicted from cryoEM images of this assembling system in presence or absence of salts: A first one, where DNA is sandwiched between NPQ+ surface and cationic lipids, the excess of DNA being assembled to concentric Lx; A second, where DNA is also sandwiched between the cationic nanoparticle surface and cationic lipids but covering partly the particle surface with planar Lx structures. This new type of complexation may be interesting for the orientation of lipoplexes in view of improving their stability in the presence of serum and of improving the transfection

amount of DNA complexed by quaternized nanoparticles with increasing concentrations of dextran sulfate. With the increase of the concentration of dextran sulfate more DNA was recovered in the supernatant (Figure 4d). The decrease in cosedimented DNA is shown in relation to the ratio of negative charges brought by the dextran sulfate to the charges provided by the present amount of DNA. Without the presence of dextran sulfate, 30% of DNA strands is complexed by the nanoparticles (r30, corresponding to a NP surface of 0.64 nm2/ bp). At a ratio of charges dextran sulfate/DNA of 0.2 we already reached 15% of cosedimentation which means that the nanoparticles preferably interact with dextran sulfate due to its higher charge density per mass unit (about 2 times more) and its higher flexibility compared to the rather rigid DNA strands. This result gives evidence for the pure electrostatic nature of the interactions between DNA and trimethylated (Me3) ammonium head groups as already previously observed without any additional hydrophobic interactions with the nonpolar groove of the helix that could be observed for example with MeBu2 and Me2Hex ones.17 Beyond the context of this study, the scope of these results can find some interest not only for DNA-transfection mediated by cationic nanoparticles11,13 but also for DNA purification processes using for example cationic magnetic nanoparticles41,42 where conditions of capture and elution have to be as efficient as possible. 3.4. Interactions of NPQ+/DNA Complexes with Cationic Lipids. In the aim to design a new DNA cargo, NPQ+/DNA complexes have to be covered by transfecting agents such as cationic lipids. In this part a (cryo-)TEM study of competitive interactions between NPQ+/DNA complexes and cationic lipids is presented. SUVs of DOTAP, lipid often used in lipofection,43 are used here for its cationic headgroup similar as the one of NPQ+ surface ligands, at the (lipid + NPQ+)/DNA charge ratio of around 10 to allow the complexation of all DNA molecules with nanoparticles and cationic lipids. Experiments have been achieved at pH 6.5 for which the ζ potential reaches an optimal value of 40 mV, in the presence of 150 mM NaCl and in the absence of salt. In presence of salt, it has been observed supported lipid bilayers (SLB) of DOTAP in suspension at a certain distance from the surface of the quaternized nanoparticles (Figure 5, parts a, b, and c; SLBs are indicated by gray arrows). This bilayer is characterized by its thickness of 3.7 nm, quite thinner than that of classical phospholipid such as 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC, 5.4 nm).44 This bilayer thinning is attributed to the Coulombic repulsions which increase the developed surface area of the TAP headgroup despite the absence of phosphatidyl moiety present in DOPC. This larger headgroup leads to bilayer compression through an increase of the hydrocarbon chain disorder and tail interdigitation.44 The formation of such “suspended” SLB of DOTAP is quite different from those already observed with lipid vesicles interacting with silica nanoparticle surface. SLBs are generally well characterized in cryo-EM by a continuous ring of electrondense material, separated from the particle edge by a distance that corresponds to the known thickness of a lipid bilayer45 around 4−5 nm depending to the nature of lipid. The outer, distal, lipid leaflet of the SLB follows faithfully the surface roughness of silica particles while the inner, proximal, lipid leaflet is not resolved due to its closeness to the particle edge, the resolution of the cryo-EM images being around 2 nm.46,47 In the present case, the double leaflet is well resolved with the inner leaflet located at most 2 nm from the surface (Figures 5b, 6409


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The Journal of Physical Chemistry B efficiency of Lx.9,51,52 They could also promote a singular endocytosis path for the transport of gene material through the cell membrane.

recipients of a Ph.D. fellowship from the French Ministry of Education and Research and Technology (MENRT).

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4. CONCLUSIONS By using an appropriate chemical surface functionalization leading to silica nanoparticles bearing quaternary ammonium groups, it was possible, in particular, to experiment a novel type of tripartite complex composed of cationic silica colloidal surfaces, cationic lipids and DNA molecules for various salt conditions. The high surface site density of quaternary ammoniums has imparted to nanoparticles an unusual electrostatic colloidal stability more particularly at high salt concentration (up to 300 mM at pH 7.4). Thanks to this property it has been shown that the DNA binding capacity of the cationic nanoparticles was enhanced for these high concentrations of salt due to the conformational change of the DNA molecules. Beyond the frame of this study, this result open the way for the development of new methods of DNA extraction and purification. The subsequent study of electrostatic interactions with cationic liposomes has led to the formation of NPQ+/DNA/lipid complexes where DNA rods are sandwiched between NP surface and SLB and where multilamellar Lx shown different orientations in presence or absence of salts. These morphologies could impact the transfection properties and stability in biological environment. For future investigations, such surface functionalization approach will be applied for different sizes of labeled silica nanoparticles. It will be then possible to track the intracellular fate of these “liponanoplexes” and to assess the in vivo and in vitro performances of such systems in terms of vectorisation and transfection efficiency with the size and the morphology of such tripartite assembled complexes.

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(1) Edelstein, M. L.; Abedi, M. R.; Wixon, J.; Edelstein, R. M. Gene Therapy Clinical Trials Worldwide 1989−2004-an Overview. J. Gene Med. 2004, 6, 597−602. (2) Vijayanathan, V.; Thomas, T.; Thomas, T. J. DNA Nanoparticles and Development of DNA Delivery Vehicles for Gene Therapy. Biochemistry 2002, 41, 14085−14094. (3) Barteau, B.; Chèvre, R.; Letrou-Bonneval, E.; Labas, R.; Lambert, O.; Pitard, B. Physicochemical Parameters of Non-Viral Vectors that Govern Transfection Efficiency. Curr. Gene Ther. 2008, 8, 313−323. (4) Lee, D.-E.; Koo, H.; Sun, I.-C.; Ryu, J. H.; Kima, K.; Kwon, I. C. Multifunctional Nanoparticles for Multimodal Imaging and Theragnosis. Chem. Soc. Rev. 2012, 41, 2656−2672. (5) Bihan, O. L.; Chèvre, R.; Mornet, S.; Garnier, B.; Pitard, B.; Lambert, O. Probing the In Vitro Mechanism of Action of Cationic Lipid/DNA Lipoplexes at a Nanometric Scale. Nucleic Acids Res. 2011, 39, 1595−1609. (6) Manning, G. S. Limiting Laws and Counterion Condensation in Colyelectrolyte Solutions. I. Colligative Properties. J. Chem. Phys. 1969, 51, 924−933. (7) Rädler, O.; Koltover, I.; Salditt, T.; Safinya, C. R. Structure of DNA-Cationic Liposome Complexes: DNA Intercalation in Multilamellar Membranes in Distinct Interhelical Packing Regimes. Science 1997, 275, 810−814. (8) Madeira, C.; Loura, L. M. S.; Aires-Barros, M. R.; Prieto, M. Fluorescence Methods for Lipoplex Characterization. Biochim. Biophys. Acta 2011, 1808, 2694−2705. (9) Chèvre, R.; Bihan, O. L.; Beilvert, F.; Chatin, B.; Barteau, B.; Mével, M.; Lambert, O.; Pitard, B. Amphiphilic Block Copolymers Enhance the Cellular Uptake of DNA Molecules Through a Facilitated Plasma Membrane Transport. Nucleic Acids Res. 2011, 39, 1610−1622. (10) Roy, I.; Ohulchanskyy, T. Y.; Bharali, D. J.; Pudavar, H. E.; Mistretta, R. A.; Kaur, N.; Prasad, P. N. Optical Tracking of Organically Modified Silica Nanoparticles as DNA Carriers: a Nonviral, Nanomedicine Approach for Gene Delivery. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 279−284. (11) Kneuer, C.; Sameti, M.; Bakowsky, U.; Schiestel, T.; Schirra, H.; Schmidt, H.; Lehr, C. M. A Nonviral DNA Delivery System Based on Surface Modified Silica-Nanoparticles Can Efficiently Transfect Cells In Vitro. Bioconjugate Chem. 2000, 11, 926−932. (12) Kneuer, C.; Sameti, M.; Haltner, E. G.; Schiestel, T.; Schirra, H.; Schmidt, H.; Lehr, C. M. Silica Nanoparticles Modified with Aminosilanes as Carriers for Plasmid DNA. Int. J. Pharm. 2000, 196, 257−261. (13) He, X.; Wang, K.; Tan, W.; Liu, B.; Lin, X.; He, C.; Li, D.; Huang, S.; Li, J. Bioconjugated Nanoparticles for DNA Protection from Cleavage. J. Am. Chem. Soc. 2003, 125, 7168−7169. (14) Kumar, M. N. V. R.; Sameti, M.; Mohapatra, S. S.; Kong, X.; Lockey, R. F.; Bakowsky, U.; Lindenblatt, G.; Schmidt, H.; Lehr, C. M. Cationic Silica Nanoparticles as Gene Carriers: Synthesis, Characterization and Transfection Efficiency In Vitro and In Vivo. J. Nanosci. Nanotechnol. 2004, 4, 876−881. (15) Ghosh, P. S.; Kim, C.-K.; Han, G.; Forbes, N. S.; Rotello, V. M. Efficient Gene Delivery Vectors by Tuning the Surface Charge Density of Amino Acid-Functionalized Gold Nanoparticles. ACS Nano 2008, 2, 2213−2218. (16) Lee, S. E.; Sasaki, D. Y.; Perroud, T. D.; Yoo, D.; Patel, K. D.; Lee, L. P. Biologically Functional Cationic Phospholipid-Gold Nanoplasmonic Carriers of RNA. J. Am. Chem. Soc. 2009, 131, 14066−14074. (17) Jin, C.; Han, L.; Che, S. Synthesis of a DNA−Silica Complex with Rare Two-Dimensional Square p4mm Symmetry. Angew. Chem., Int. Ed. 2009, 48, 9268−9272. (18) Goodman, C. M.; Rotello, V. M. DNA-Binding by Functionalized Gold Nanoparticles: Mechanism and Structural Requirements. Chem. Biol. Drug Des. 2006, 67, 297−304.

ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of colloidal silica (Figure S1), EDPS modified nanoparticles (Figure S2), alkylated EDPS-modified nanoparticles (Figure S3) and COSY 2D NMR spectrum of the same sample (Figure S4); conductometric titrations (Figure S5); procedure for herring sperm DNA purification with the electrophoresis gel (Section S6); additional cryo-EM image of NP/lipoplex agglomerations at high magnification (Figure S7); and TEM images of NP/lipid mixtures with and without DNA molecules and/or NaCl (Figure S8). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b01834.

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AUTHOR INFORMATION

Corresponding Author

*(S.M.) Telephone: +33 540 006 335. E-mail: [email protected]. Author Contributions ∥

REFERENCES

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors also acknowledge Dr. Christophe Schatz and Dr. Anne Laure Wirotius from LCPO (UMR5629 CNRSUniversity of Bordeaux-IPB) for technical assistance and helpful discussions on 1H NMR. N.R. and L.A. were each 6410


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Quaternary Ammonium Groups Exposed at the Surface of Silica

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