antisense oligonucleotide complexes

Accepted Manuscript Title: Investigation of the structural organization of cationic nanoemulsion/antisense oligonucleotide complexes Author: Fernanda Bruxel Jos´e Mario Carneiro Vilela ˆ Margareth Spangler Andrade Angelo Malachias Carlos A. Perez Rog´erio Magalh˜aes-Paniago Mˆonica Cristina Oliveira Helder F. Teixeira PII: DOI: Reference:

S0927-7765(13)00552-3 http://dx.doi.org/doi:10.1016/j.colsurfb.2013.08.035 COLSUB 5985

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

28-2-2013 21-8-2013 22-8-2013

ˆ Please cite this article as: F. Bruxel, J.M.C. Vilela, M.S. Andrade, A. Malachias, C.A. Perez, R. Magalh˜aes-Paniago, M.C. Oliveira, H.F. Teixeira, Investigation of the structural organization of cationic nanoemulsion/antisense oligonucleotide complexes, Colloids and Surfaces B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.08.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Investigation of the structural organization of cationic nanoemulsion/antisense

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oligonucleotide complexes

3 Fernanda Bruxel a, José Mario Carneiro Vilela b, Margareth Spangler Andrade b, Ângelo

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Malachias c, Carlos A. Perez d, Rogério Magalhães-Paniago c, Mônica Cristina Oliveira e, and

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Helder F. Teixeira a*

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a

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Rio Grande do Sul (UFRGS), Av. Ipiranga 2752, 90610-000 Porto Alegre, Brazil.

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Programa de Pós-graduação em Ciências Farmacêuticas da Universidade Federal do

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b

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Silveira, 2000, 31170-000 Belo Horizonte, Brazil

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c

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6627, 31270-901 Belo Horizonte, Brazil

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d

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13083-970 Campinas, São Paulo, Brazil

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Departamento de Física, Universidade Federal de Minas Gerais, Av. Antônio Carlos,

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Laboratório Nacional de Luz Síncrotron, Rua Giuseppe Máximo Scolfar, 10000,

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Fundação Centro Tecnológico de Minas Gerais – CETEC, Avenida José Candido da

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Laboratório de Farmacotécnica e Tecnologia Farmacêutica, Departamento de Produtos

Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte, Brazil

* Address for correspondence: Faculdade de Farmácia - Universidade Federal do Rio Grande do Sul Av. Ipiranga, 2752, 90610-000, Porto Alegre, RS, Brazil Tel.: +55-51-33165090 Fax: +55-51-33165437 [email protected]

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ABSTRACT

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Atomic force microscopy image analysis and energy dispersive x-ray diffraction experiments

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were used to investigate the structural organization of cationic nanoemulsion/oligonucleotide

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complexes. Oligonucleotides targeting topoisomerase II gene were adsorbed on cationic

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nanoemulsions obtained by means of spontaneous emulsification procedure. Topographical

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analysis by atomic force microscopy allowed the observation of the nanoemulsion/

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oligonucleotide complexes through three-dimensional high-resolution images. Flattening of

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the oil droplets was observed, which was reduced in the complexes obtained at high amount

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of adsorbed oligonucleotides. In such conditions, complexes exibit droplet size in the 600 nm

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range. The oligonucleotides molecules were detected on the surface of the droplets,

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preventing their fusion during aggregation. A lamellar structure organization was identified

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by energy dispersive x-ray diffraction experiments. The presence of the nucleic acid

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molecules led to a disorganization of the lipid arrangement and an expansion in the lattice

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spacing, which was proportional to the amount of oligonucleotides added.

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KEYWORDS

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Antisense Oligonucleotides; Atomic force microscopy; Cationic lipids; Nanoemulsions,

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Energy dispersive X-ray diffraction; Phospholipids; X-ray diffractometry.

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INTRODUCTION

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Nucleic acids based therapy has been considered as a promising strategy for inherited and

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acquired diseases [1]. The down-regulation at the post-transcriptional level may be achieved

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by small nucleic acids, including antisense oligodeoxynucleotides (ON), which are able to

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recognize the target messenger RNA through a specific base pairing process [2]. However,

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ON-based therapy is limited by the rapid degradation of ONs in biological fluids and the

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inability to cross cell membranes, due to their hydrophilic character and large molecular

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structure [3].

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There has been considerable progress in developing nanostructured systems for the delivery

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of ON [4]. Among them, the use of cationic nanoemulsions has been described as a promising

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strategy to improve nucleic acids transfection to mammalian cells, protecting them against

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nuclease attack [5, 6]. ON molecules can be associated with oil droplets through ion-pair

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formation due to the positively-charged nanoemulsions and the negatively-charged ON. The

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extent of ON attraction was found to be dependent on the cationic lipid (nature of the cationic

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polar head group and the acyl chains) and the ON structures (lengths and base composition)

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[7-9]. Since ON may form alternative secondary or tertiary structures within themselves [10],

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the characterization of the ON interactions with cationic nanoemulsions is a key

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consideration. Such interactions may play a crucial role in their association and release

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kinetics [11-13].

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Recent reports in the literature have showed that ON targeting topoisomerase II of the parasite

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seems to be an interesting target for antisense therapy against malaria [14, 15]. In this context,

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we have recently demonstrated that a specific ON sequence against the messenger RNA of

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that enzyme can be efficiently adsorbed in an optimized cationic nanoemulsion by means of a

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full-factorial design [11]. ON-loaded nanoemulsions were found to be located inside the

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infected erythrocytes, inhibiting parasite growth (up to 80%) and causing a delay in the P.

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falciparum life cycle [16].

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Despite the potential for cationic nanoemulsions as ON delivery systems, the characterization

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of the complexes still remains poorly studied and understood. While there are some reports

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concerning supramolecular organization of ON associated with cationic liposomes [17, 18],

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none has dealt with the association of ON with cationic nanoemulsions. Besides, it is well

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described that the lipid structure and morphology of lipoplexes have an impact on the

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transfection efficiency [19]. Thus, in this study, the interactions between cationic

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nanoemulsions and either phosphodiester or phosphorothioate ON were investigated by

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atomic force microscopy, and energy dispersive X-ray diffraction.

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EXPERIMENTAL

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Chemical and reagents

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Medium-chain triglycerides, egg-lecithin, and dioleoyltrimethylammoniumpropane (DOTAP)

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were purchased from Lipoid AG (Germany), glycerol from Merck (Brazil) and ethanol from

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Quimex Ind. Químicas (Brazil). The antisense phosphodiester (PO, MW = 9146 g/mol) and

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phosphorothioate (PS, MW = 9610 g/mol) oligonucleotide sequences (5’ ATG TAA TAT

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TCT TTT GAA CCA TAC GAT TCT 3’) were purchased from Invitrogen Brazil Ltda.

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Preparation and characterization of nanoemulsions/oligonucleotide complexes

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Nanoemulsions (NE) were obtained by means of the spontaneous emulsification procedure

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[20]. The final composition (%, w/w) was medium chain triglycerides 8.0, egg-lecithin 2.0,

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glycerol 2.25, DOTAP 0.132 and MilliQ® water up to 100. A control formulation obtained in

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the absence of the cationic lipid DOTAP was also prepared (lecithin-formulation).

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ON association was performed at the end of the manufacturing process. PO or PS water

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solutions were added to the cationic NE and incubated for 15 minutes at room temperature,

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resulting in NE/PO and NE/PS complexes. They were prepared at +4/- and +0.2/- charge

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ratios. This +/- charge ratio is the theoretical ratio between the number of positive charges

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from the cationic lipid in NE, and the number of negative charges from the phosphate groups

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of the ON. The ON association was estimated by the ultrafiltration/centrifugation procedure

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[20]. After separating a portion of the water phase using regenerated cellulose membranes at a

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30 kDa cut-off (Millipore, USA), free ON was determined in the clear ultrafiltrate by

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spectrophotometry at 262 nm (spectrophotometer Hewlett-Packard 8452A).

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Size distribution and zeta potential of NE, NE/PO, and NE/PS were determined through

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photon correlation spectroscopy (PCS, 25°C, 90° angle) and Laser Doppler electrophoresis,

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respectively, in a Malvern Zetasizer Nano ZS (Malvern Instrument, UK). The samples were

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adequately diluted in 1 mM NaCl solution for the measurements.

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Atomic force microscopy (AFM)

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A droplet of approximately 10 μL from each sample (NE, PO, PS, NE/PO, and NE/PS) was

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deposited on a freshly cleaved mica surface and dried with an argon stream. All

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measurements were carried out in air and at room temperature, immediately after deposition

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on a mica surface. The tapping mode was used to avoid sample damage. Silicon probes

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(NanosensorsTM) were used, with 228 μm long cantilevers, with 75-98 kHz frequencies. The

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spring constants were 3–7 N/m or 29–61 N/m, the nominal tip curvature radius 5–10 nm and

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the scan rate 1 Hz. The diameter/height ratios were calculated after measuring of 40 droplets.

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AFM experiments were performed using a Dimension 3100 apparatus with a Nanoscope IIIa

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controller (Digital Instruments, Santa Barbara, CA, USA).

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Energy dispersive X-ray diffraction (EDXD)

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Experiments were performed using a setup built at the X-Ray Fluorescence beamline (D09B–

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XRF) [21] of Brazilian Synchrotron Light Laboratory (LNLS, Campinas, SP), yielding a

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bandpass between 4 and 25 keV. EDXD patterns were detected by a solid state detector

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(scattering angle of 2θ = 1.4o) with an energy resolution of 165 eV within a q range of 0.10–

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0.60 Å-1. The exposure time and synchrotron current were used to normalize the

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diffractogram. Samples were deposited directly on a clean silicon surface and allowed to dry

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prior to measurements. Experiments were performed for NE before and after complexation

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with PO and PS at +4/- and +0.2/- charge ratios. The EDXD profile was also obtained for the

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control of lecithin formulation. Exposure time varied from 100 to 300s as no sample damage

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was observed during this period.

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RESULTS AND DISCUSSION

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AFM is a powerful technique for investigating the structure of a wide variety of nanoscale

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objects, including biological molecules and lipid systems under aqueous environments [22].

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AFM images captured in ‘‘height’’ mode enable us to see the topography of the blank

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nanoemulsion oil droplets. Images showed individual droplets heterogeneously distributed in

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terms of size and height, with a mean droplet size of approximately 600 nm (Figure 1A).

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The diameter/height ratios were calculated using the topography section analysis since we

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could not derive the horizontal diameter from the vertical dimension of the droplets, as

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previously described for other colloidal carriers, such as parenteral emulsions [23], liposomes

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[24], or nanocapsules [25, 26]. The diameter/height ratio of cationic nanoemulsion droplets

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indicated a diameter value more than 20 times their height. Some flat structures consisting of

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several layers could be observed.

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The antisense PO and PS oligonucleotides were added to blank NE at two different charge

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ratios (+4/- and +0.2/-). The complexation of PO and PS was evidenced by the

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reduction/inversion of zeta potential from +47.9 ± 3.2 mV (blank NE) to +36.6 ± 1.9 mV

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(+4/- NE/PO), +33.8 ± 5.3 mV (+4/- NE/PS), -20.5 ± 0.7 mV (+0.2/- NE/PO) and -22.5 ± 2.3

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mV (+0.2/- NE/PS). In addition, free oligonucleotides were not detected in the ultrafiltrate

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after ultrafiltration/centrifugation of complexes.

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AFM images of the complexes were presented in figures 1B and 1C, respectively. For +4/-

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complexes, the measured diameter of droplets remained approximately 600 nm. However, in

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the presence of high concentrations of ON (+0.2/- charge ratio), the average droplet diameter

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was around 200 to 300 nm. For such complexes, less flattened droplets were seen, and a

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calculated diameter/height ratio of 10 was obtained. The flattening process was ascribed to

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different reasons, including the pressure applied by the tip during scanning [23, 27] and the

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embedding of the sample dispersion due to drying on mica [23]. Such reasons may contribute

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to explaining the much higher droplet size observed in AFM experiments when compared

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with the PCS ones (from 186 ± 23 to 244 ± 17 nm). Our results also showed the effect of the

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ON concentration on the flattening phenomenon and the diameter/height ratio value. NE/ON

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complexes obtained at low ON content (+4/- charge ratio) exhibited diameter/height values

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near to 20. However, the flattening process was reduced in the presence of higher amount of

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ON adsorbed on NE (+0.2/- charge ratio), reaching values of approximately 10. Such

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diameter/height value data are described for “more rigid” carriers, such as nanocapsules [23,

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25, 27].

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To observe the droplet surface of NE/ON complexes, AFM images were additionally captured

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in phase contrast mode (Figure 2). The deposition of free ON at different concentrations on a

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mica surface was first evaluated for comparison purposes (Figure 2A). No previous treatment

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on mica was necessary since the analysis was performed in air just after sample preparation.

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When ON were deposited on untreated freshly cleaved mica, it was possible to see

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agglomerated structures, homogenously distributed on the surface. The DNA molecules are

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transported to the surface by diffusion and equilibrate onto the surface as in an ideal two-

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dimensional solution [28, 29]. ON molecules spontaneously self-organized as a uniform

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network on the mica surface, revealing a vertical roughness from 0.8 to 1.2 nm. Similar

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observations were reported for ssDNA oligomers on gold substrates and on untreated highly

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oriented pyrolytic graphite [30, 31]. Moreover, the deposition of a highly concentrated ON

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solution leads to a denser network formation, which was not complete, presenting some holes

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on it (data not shown).

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Images of free ON were compared to those of the cationic NE/ON complexes. Considering

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phase contrast images of the droplets of NE/ON complex (Figure 2B), we observed a

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contrasting surface, which was very similar to that observed for the free ON network (Figure

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2A). It is important to emphasize that these contrasting phase images could not be seen for

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blank NE (Figure 3D), as well as for other regions than NE/ON complex droplets, when

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reducing the z-scale in the microscopic field of Figure 2B. These findings suggest the location

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of the ON molecules on the surface of oil droplets. In addition, some clusters were observed

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in blank NE and NE/ON complexes. Interestingly, for +0.2/- NE/ON complexes, even when

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agglomerated, the droplets of the clusters were not completely fused, but rather standing next

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to one another (Figure 2C). As for free ON, a very similar phase contrast image was observed

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on these agglomerates. We may then suppose that ON could be acting as a protective coating

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for NE, preventing complete aggregation of the oil droplets.

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The EDXD technique has been applied to various biological compounds and it is very helpful

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in studying the structural organization of nucleic acids/cationic carrier complexes [32-34].

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EDXD diffraction patterns of blank NE are presented in figure 3. As can be seen, well-defined

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peaks were observed, with Bragg reflections of q = 0.099, 0.195, and 0.283 Å-1, showing a

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lamellar organization (Figure 3B). Two small extra peaks were observed at q = 0.118, 0.127

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Å-1 for this sample. For the lecithin-formulation, obtained in the absence of the cationic lipid

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DOTAP, two broad peaks appeared at q = 0.128 and 0.245 Å-1 (Figure 3A), corresponding, to

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the first and second order of a lamellar periodic structure.

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Concerning cationic NE, from the periodicities of Bragg reflections calculated by the formula

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d =2π/q, using the q-positions of the observed peaks, a lattice spacing equivalent to 63.1Å,

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32.2Å, and 22.1Å was observed (Figure 3B). This pattern of Bragg reflections, exhibiting

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ratios equal to 1, 2, and 3, corresponds to a lamellar structure organization of the lipid

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dispersed system. The lattice spacing of 63 Å corresponds to the distance of a lamellar

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periodicity, which is likely due to the binary mixture of cationic lipid DOTAP and

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phospholipids from egg-lecithin. The structural dimension of the lamellar phase is in

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agreement with the data reported in other studies for DOTAP liposomes (63.5 Å) [35] and

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nanoemulsions (69 Å) [36] by small-angle X-ray diffraction. The two small extra peaks

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observed (that correspond to real space distances of approximately 49-52 Å) could be

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attributed to different domains of the lipid layers, considering the presence of different

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phospholipids of egg-lecithin, since they were also observed in the EDXD diffractograms of

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control lecithin-nanoemulsions (Figure 3A). In addition, this control lipid system, obtained in

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the absence of DOTAP, was found to be less ordered when compared to the cationic ones,

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since much broader peaks of the first and second order Bragg reflections were observed. This

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suggests that the presence of DOTAP may organize the phospholipid molecules. We may

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suppose that the lamellar structure is due to the deposition of lipid layers on the surface of

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droplets of the NE. This organization may occur as a result of the presence of excess of

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phospholipids in the formulation, as we previously discussed in other study [8]. The lattice

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spacing of 63 Å may, therefore, be related to the superposition of lipid layers on the polar

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headgroup of phospholipids, whose lipophilic chains are inserted in the oil core of the

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nanoemulsions.

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Figure 4 shows EDXD diffraction patterns of NE/ON complexes containing either PO or PS.

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For NE/PO complexes prepared in the charge ratio of +4/- and +0.2/-, Bragg reflections at q =

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0.099, 0.191, and 0.278 Å-1 (Figure 4A) and q = 0.083, and 0.165 Å-1 (Figure 4B) were

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observed, respectively. For NE/PS complexes, Bragg reflections at q = 0.092, and 0.161 Å-1

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(Figure 4C) and q = 0.076, and 0.162 Å-1 (Figure 4D) were observed for complexes prepared

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in the charge ratio of +4/- and +0.2/-, respectively.

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As well as for blank NE, the diffraction patterns also indicated the existence of a lamellar

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periodic organization for all studied complexes. However, ON complexation with cationic NE

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provoked the disorganization of the lipid arrangement. Both NE/PO and NE/PS complexes

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showed a broadening of the diffraction peaks. This enlargement of peaks occurred at +4/- and

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+0.2/- charge ratio, being higher in the latter case (Figure 4B and 4D). It is interesting to note

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that the high amount of PO and PS (+0.2/- charge ratio) also induced an increase in the

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lamellar periodicity when compared with cationic NE without ON (75.7 Å, 82.7 Å and 63 Å,

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respectively). Therefore, these findings lead us to suppose that ON may be inserted in the

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lamellar arrangement, expanding the lattice spacing at high concentrations. Similar

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observations were found with the DOTAP/ON system in which SAXS studies showed an

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aqueous layer thickness of 11.8 Å, sufficient for a monolayer of ON [37]. These authors

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proposed that ON molecules may be able to penetrate into the headgroup region of the

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bilayers, contributing to the interlamellar spacing of the DOTAP/ON system. In our studies,

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we found an expansion in lattice spacing between 12.7 to 19.7 Å, which may be compatible

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with the location of ON molecules along the lipid layers.

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CONCLUSION

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This study offered a first insight in understanding the interactions of the cationic lipid

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nanoemulsions with small single strand DNA molecules. It was possible to demonstrate the

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existence of interactions between phosphodiester and phosphorothioate ON and cationic

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nanoemulsions by identifying changes in the physicochemical properties of nanoemulsions at

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the supramolecular level.

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ACKNOWLEDGEMENTS

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The authors wish to thank CAPES/COFECUB (540/06) for their financial support, and

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Laboratório Nacional de Luz Síncrotron (LNLS) for the EDXD analysis.

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microscopy and ultrasonic resonator technology on nanoscale: distinction of nanoemulsions

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D.C. Reimer, V.P. Pozharov and

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delivery of siRNA, antisense oligonucleotides and anticancer drug, Pharm. Res., 26 (2009)

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382-394.

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ovarian cancer cells, Eur. Phys. J. E Soft Matter, 10 (2003) 331-336.

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structure and transfection efficiency: a study of DC-Chol--DOPE/DNA complexes, Colloids

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FIGURE CAPTION

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Figure 1. Atomic force microscopy images of cationic nanoemulsions (A), cationic

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nanoemulsions/antisense oligonucleotides complexes in the charge ratio of +4/- (B) and

4

nanoemulsions/antisense oligonucleotides complexes in the charge ratio of +0.2/- with the

5

corresponding section analysis (C). Images are representative of the analysis of three different

6

samples (n = 3).

7

Figure 2. Atomic force microscopy images of height (left) and phase (right) of antisense

8

oligonucleotides (A), +0.2/- cationic nanoemulsions/antisense oligonucleotides complexes (B

9

and C) and cationic nanoemulsions (D) deposed on freshly cleaved mica. Images are

an

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1

representative of the analysis of three different samples (n = 3).

11

Figure 3. EDXD spectra of control lecithin-nanoemulsions (A) and cationic nanoemulsions

12

(B). The inserts show the same curves in logarithmic scattering intensity scale. The presented

13

EDXD spectra is representative of the analysis of three different samples (n = 3).

14

Figure 4. EDXD spectra of cationic nanoemulsions/phosphodiesther oligonucleotides in the

15

charge ratio of +4/- (A) or +0.2/- (B), and cationic nanoemulsions/phosphorothioate

16

oligonucleotides in the charge ratio of +4/- (C) or +0.2/- (D). In the insert, logarithmic scale

17

scattering profiles. The presented EDXD spectra is representative of the analysis of three

18

different samples (n = 3).

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19 20

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ip t cr us an d

M 3

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2

[Figure 1]

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ip t cr us an M d te Ac ce p 1 2

[Figure 2] 19 Page 19 of 22

4

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3

[Figure 3]

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ip t cr us an M

Oligonucleotides

A75.7 Å Intensity (ar b. units)

100000

105000

[Figure 4]

10000

1000

Phospholipids and cationic lipid

Ac ce p

Intensity (arb. units)

2 140000 3

te

175000

d

1

100

0,05 0,10 0,15 0,20 0,25 0,30 0,35 -1

q[Å ]

70000

35000 0 0,05

0,10

0,15

0,20

0,25

0,30

0,35

-1

q[Å ]

Structural organization of antisense oligonucleotide molecules on the oil/water interface of cationic nanoemulsions, as suggested by energy dispersive X-ray diffraction and atomic force microscopy studies.

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HIGHLIGHTS

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Cationic nanoemulsions presented dimension of a lamellar structure organization.

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Oligonucleotide molecules led to a disorganization of the lipid arrangement.

4

A flattening phenomenon of emulsion droplets was observed by AFM experiments.

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Oligonucleotides molecules on the surface of emulsion droplets prevent their fusion.

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