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Nanostructures of cationic amphiphilic cyclodextrin complexes with DNA Valentina Villari, Antonino Mazzaglia, Raphael Darcy, Caitriona M O'Driscoll, and Norberto Micali Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm3018609 • Publication Date (Web): 18 Feb 2013 Downloaded from http://pubs.acs.org on February 19, 2013
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Nanostructures of cationic amphiphilic cyclodextrin complexes with DNA Valentina Villari,∗,† Antonino Mazzaglia,∗,‡ Raphael Darcy,¶ Caitriona M. O’Driscoll,§ and Norberto Micali† CNR-IPCF Istituto per i Processi Chimico-Fisici, Viale F. Stagno d’Alcontres 37, I-98158, Messina, Italy, CNR-ISMN Istituto per lo Studio dei Materiali Nanostrutturati, Viale F. Stagno d’Alcontres 31, I-98166, Messina, Italy, School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland, and School of Pharmacy, University College Cork, Cork, Ireland E-mail:
[email protected];
[email protected] Abstract Complexes of cationic amphiphilic cyclodextrins heptakis[2-(ω -amino-oligo(ethylene glycol))6-deoxy-6-hexadecylthio]-β -cyclodextrin and heptakis[2-(ω -amino-oligo(ethylene glycol))-6deoxy-6-dodecylthio]-β -cyclodextrin with DNA were examined by small-angle X-ray scattering and dynamic as well as electrophoretic light scattering. The first cyclodextrin forms bilayer vesicles in water which, in the presence of calf thymus DNA, transform to a multilamellar complex. In this complex the DNA lies between the two polar layers of the cyclodextrin’s protonated amino groups in alternation with the lipidic bilayers. The cyclodextrin with shorter lipid chains, in contrast, forms micelles in water, and electrostatic clustering of these about DNA does not affect their intrinsic structure. These results are relevant to the potential of such ∗ To
whom correspondence should be addressed
† CNR-IPCF ‡ CNR-ISMN ¶ Univ § Univ
Coll Dublin Coll Cork
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cyclodextrins in therapeutic gene delivery, showing that their self-assembly modes in isolation influence their complex formation with DNA and possibly their efficiency in promoting cell transfection.
Introduction Complexes of cationic lipids and cationic amphiphiles with DNA have been central to the anticipated development of therapeutic non-viral gene delivery. 1,2 The two typical features of these molecules, namely cationic charge and lipophilicity, make possible the compaction of DNA by neutralisation of the anionic charges on this polyelectrolyte, followed by lipidic interaction with the bilayer cell membrane leading to cell entry by endocytosis. Since the discovery of gene silencing these molecular talents have also been used in research on the delivery of RNA. 3,4 The structures of complexes formed by cationic lipids with DNA have been shown by small angle X-ray scattering to involve multilamellar arrays in which DNA alternates with lipid bilayers. 5–7 While polycationic polymers 8 have also been much used for research into the same applications, the success of these delivery vectors is based mainly on the property which they share with cationic lipids, of forming cationic nanoparticles that adhere to cell surfaces. Cell adhesion however is indiscriminate and cytotoxic, yet the prospect of overcoming this drawback for lipids, while maintaining their lipidic cell wall fusion, makes their interactions with DNA and siRNA particularly interesting. Amphiphilic cyclodextrins incorporate lipid groups, and the assemblies formed by these mesomolecular amphiphiles include micelles and vesicles. 9 Research on these has concentrated on their application as drug delivery systems based on their assembly properties, 10–17 and includes studies of recognition and cluster effects in receptor targeting, 18–21 of inclusion of guest molecules and their transport 22 and of photosensitiser transport for photodynamic therapy. 23,24 Some of us showed 25,26 that polycationic versions of the amphiphilic cyclodextrins are effective in cell-delivery of plasmid DNA and amphiphilic cationic cyclodextrins have by now been developed
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as a distinct and important class of DNA 27–33 and siRNA 34 delivery vector. But control of the DNA compaction, and correlation between the amphiphilic cyclodextrin self-assembled structures and effectiveness of delivery, are still under investigation. These cationic amphiphilic cyclodextrins induces DNA compaction unlike regular cyclodextrins, which were proved to form inclusion complexes with surfactants/lipids and to cause the DNA decompaction in solution. 35,36 Here we have investigated structural features of the complexes formed by DNA with, for the first time, cationic amphiphilic cyclodextrins, one of which has been used for cell transfection. 25 These cyclodextrins, heptakis[2-(ω -amino-oligo(ethylene glycol))-6-deoxy-6-hexadecylthio]-β -cyclodextrin and heptakis[2-(ω -amino-oligo(ethylene glycol))-6-deoxy-6-dodecylthio]-β -cyclodextrin (Scheme 1 1-2) have hydrophobic thioalkyl chains at the 6-positions of the glucose units, while the polar groups at the 2-positions are short oligo(ethylene oxide) chains (PEG≈120 ). These terminate in amino groups which confer polycationic properties in water, while in the case of the neutral cyclodextrin 3 they terminate in hydroxyl groups. The investigation has been carried out by small angle X-ray scattering (SAXS) and by dynamic and electrophoretic light scattering, in order to access morphological information on the CD.DNA complexes and on the location of DNA in the complexes.
Scheme 1: Structures of investigated amphiphilic cyclodextrins.
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Experimental Material SC16NH2, SC12NH2 and SC16OH were synthesised according to the procedures previously described elsewhere. 13 Colloidal stock dispersions of each cyclodextrin in water (SC16NH2 0.5 mg/ml, SC12NH2 0.7 mg/ml, SC16OH 0.4 mg/ml) were prepared by dissolution in methylene chloride, evaporation overnight and hydration with ultrapure water (Galenica Senese) for 1 h. The colloidal dispersion was sonicated for 3 h at 50o C. Calf thymus DNA solution (ctDNA, Type I fibres from Sigma, 0.6 mg/ml) was prepared in ultrapure water and stirred overnight at room temperature. According to a recent work by Porsch et al. 37 this ctDNA has a mass-weighted average number of base-pairs of about 13000. pCMS-EGFP plasmid DNA (pDNA, 5.5 kb, Clontech, Palo Alto, CA) 38,39 was amplified by means of cultured E.coli XL 1-Blue cells. pDNA solution (0.6 mg/ml) was prepared similarly to ctDNA. The concentration and purity of ctDNA and pDNA solutions were established by UV spectrophotometry and gel-electrophoresis. Colloidal dispersions of cyclodextrin (CD) and of complexes with ctDNA (CD.ctDNA) were prepared at CD concentrations 0.1 and 0.3 mg/ml and at mass ratios (MR CD:ctDNA=4, 10, 18). The complex CD.pDNA was prepared at CD concentrations 0.1 mg/ml and MR CD:pDNA=10. When referring to the complexes, we preferred to use the mass ratio, instead of the charge ratio, because the hydrolysis equilibrium (pKh ) of ammonium end-groups of the oligo(ethylene glycol) chains, is responsible for the partial protonation of the amphiphilic cyclodextrin as it will be shown. The pH of the CD/DNA solutions was in the range 6.4-7.0, as measured by pH-Meter-Metrohm 744, for mass ratio changing between 18 and 4.
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Small Angle X-ray Scattering (SAXS) The Small Angle X-ray measurements were carried out by using the high brilliance ID02 beamline of the ESRF (Grenoble, France). The incident beam energy was 12.5 keV and the two sampledetector distances adopted were 1 and 5 m (obtaining a Q range from 0.02 to 5 nm−1 ). Different images with acquisition time of 50 ms each were taken in a Peltier controlled flow-through cell (quartz capillary thickness 1.8 mm) in order to measure always fresh solution and avoid radiation damage. This cell also allowed for reliable solvent subtraction. The temperature was set at 25 and 40o C and the measurements on each sample was performed twice. Data reduction was performed by using the ID02 free software package (SAXSutilities). The absolute excess scattered intensity is: 40,41 I(Q) ∝ P(Q)S(Q)
(1)
where P(Q) and S(Q) are the normalized form factor and the structure factor, respectively and Q is the exchanged wave vector. For dilute solutions, as those investigated, interparticle interactions can be neglected (S(Q) ≈ 1), so that the scattered intensity is directly related to the form factor of the scattering object. The fit of the scattered intensity was performed by using different models. One is that of a bilayer with a symmetric electron density profile constituted by two Gaussians for the outer and inner hydrophilic shells and one for the hydrophobic central shell, according to the law: 42,43 2 P(Q) = Q−2 2σH exp −σH2 Q2 /2 cos (QzH ) − σC ρr exp −σC2 Q2 /2
(2)
where σH and σC control the Gaussian widths of the hydrophilic and hydrophobic region, respectively, ±zH the center of the hydrophilic regions and ρr the ratio between the electron density of the hydrophobic (ρC − ρ0 ) and hydrophilic region (ρH − ρ0 ), as sketched in Figure 1 (ρ0 being the electron density of water).
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The other model is that of a core-shell spherical micelle:
Figure 1: Electron density profile of the bilayer.
P(Q) ∝
R3o (ρH
3J1 (QRo ) 3J1 (QRi ) − R3i (ρH − ρC ) − ρ0 ) QRo QRi
2
(3)
Ro and Ri being the outer and inner radius, respectively, and J1 (QR) = [sin(QR) − QRcos(QR)]/(QR)2.
Dynamic Light Scattering (DLS) Dynamic light scattering experiments were performed by using a He-Ne laser source (λ = 633nm) at a power of 10mW, linearly polarized orthogonal to the scattering plane, and a homemade computer controlled apparatus for collecting the scattered light within an angular range 30o -150o . 44 The scattered light was analysed by a MALVERN 4700 correlator to obtain the normalized scattered electric field autocorrelation function, g1 (Q,t), where Q = (4π n/λ ) sin (θ /2) is the modulus of the exchanged wave vector (θ being the scattering angle, n the refractive index of the medium and λ the wavelength of light in vacuum). For diffusing monodisperse spherical scatterers with radius R, the normalized field autocorrelation function takes a simple exponential form, g1 (Q,t) = exp (−Γt). Under the condition QR 1) and 1 in the Hückel limit (κ R
