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Characterization of PEI-coated superparamagnetic iron oxide nanoparticles for transfection: Size distribution, colloidal properties and DNA interaction
ARTICLE IN PRESS
Journal of Magnetism and Magnetic Materials 311 (2007) 300–305 www.elsevier.com/locate/jmmm
Characterization of PEI-coated superparamagnetic iron oxide nanoparticles for transfection: Size distribution, colloidal properties and DNA interaction Benedikt Steitza, Heinrich Hofmanna, Sarah W. Kamaub, Paul O. Hassab, Michael O. Hottigerb, Brigitte von Rechenbergc, Magarethe Hofmann-Amtenbrinkd, Alke Petri-Finka, a
Laboratory of Powder Technology, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Lausanne, Switzerland b Institute of Veterinary Biochemistry and Molecular Biology, University of Zu¨rich, Zu¨rich, Switzerland c Musculoskeletal Research Unit, Equine Hospital, Vetsuisse Faculty Zurich, University of Zurich, Winterthurerstr. 260, 8057 Zurich, Switzerland d MatSearch, Chemin Jean Pavillard 14, 1009 Pully, Switzerland Available online 20 December 2006
Abstract Superparamagnetic iron oxide nanoparticles (SPIONs) were coated with polyethylenimine. Here, we briefly describe the synthesis as well as DNA:PEI:SPION complexes and the characterization of the compounds according to their particle size, z-potential, morphology, DNA complexing ability, magnetic sedimentation, and colloidal stability. PEI coating of SPIONs led to colloidally stable beads even in high salt concentrations over a wide pH range. DNA plasmids and PCR products encoding for green fluorescent protein were associated with the described beads. The complexes were added to cells and exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency. r 2006 Elsevier B.V. All rights reserved. Keywords: Superparamagnetism; Iron oxide; Transfection; Gene delivery; Colloidal stability
1. Introduction The possible applications of superparamagnetic iron oxide nanoparticles (SPIONs) are manifold ranging from various separation techniques and MRI contrast agents to drug delivery systems, magnetic hyperthermia, and magnetically assisted transfection [1]. Different coatings such as phospholipids, fatty acids, surfactants, silica, polymers, polysaccharides, and peptides of superparamagnetic nanoparticles are reported in the literature [2–4]. Various gene delivery vectors have been investigated which can be divided into two main groups: biological vectors where the gene of interest is cloned into the virus genome, and techniques employing either chemical or physical approaches such as cationic molecular carriers Corresponding author. Tel.: +41 21 693 5107; fax: +41 21 693 3089.
E-mail address: alke.fink@epfl.ch (A. Petri-Fink). 0304-8853/$ - see front matter r 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2006.10.1194
that form electrostatic interactions with DNA [5]. Magnetically assisted transfection now combines superparamagnetic nanoparticles with DNA. Plank et al. [6] have studied this method extensively. A force parallel to the field gradient acts on the magnetized particles and due to this magnetic field gradient the particles sediment on the cell surface [7]. The potential of magnetically assisted transfection might be seen in the efficient and fast delivery of genetic materials into the cells nucleus and the enhancement of gene transfection in vivo and in vitro with minimal toxicity to the cells. One important factor influencing the behaviour of the transfection agent is the size of the complexes. The size of nanoparticles has been shown to influence the rate of their uptake as well as their cytoxicity. Commercial products such as transMAGPEI (Chemicell, Germany) are relatively large particles with a size of 200 nm [7]. Rejman et al. [8] showed that nanoparticles with a size of 50 nm are
ARTICLE IN PRESS B. Steitz et al. / Journal of Magnetism and Magnetic Materials 311 (2007) 300–305
taken up 3–4 times faster than 100 nm particles and 8–10 times faster than 200 and 500 nm particles. Endocytosis is not only more efficient with 50 nm particles, but the velocity of cytoplasmatic movement was also found to be a function of particle size [9]. Estimated from the Stoke– Einstein relation, the diffusion coefficient of 50 nm nanoparticles compared to 200 nm particles is 4 times larger. In a previous study, the influence of the size of nickel ferrite nanoparticles on the cytotoxicity was determined [10]. In this study, 10 nm nanoparticles were compared to 150 nm particles, the latter showing a higher cytotoxicity than the 10 nm particles. In the present study, the characteristics of SPIONs coated with polyethylenimine (PEI) are investigated and first results about the applicability as transfection agents are reported. PEI is known to form cationic complexes, which interact non-specifically with negatively charged species such as DNA and enter the cell via endocytosis [11]. PEI offers a high positive charge density and exhibits a strong proton buffer capacity over a broad pH range. The latter characteristic is advantageous for PEI since the proton sponge effect allows sufficient gene escape without the necessity of adding endosome disrupting reagents, such as chloroquine, poly(propylacrylic acid), or peptides. Here, we briefly describe the synthesis of polymer coated SPIONs as well as SPION–PEI–DNA complexes and the characterization of the compounds with respect to their particle size, x-potential, morphology, DNA complexing ability, magnetic sedimentation, and colloidal stability, all with regard to their applicability as non-viral gene vectors. 2. Materials and methods All chemicals were of analytical reagent grade and used without further purification. Ultra-pure deionized water (Seralpur delta UV/UF setting, 0.055 mS/cm) was used in all synthesis steps. Dialysis tubing with a molecular weight cut-off at 12,000 was used for dialysis (D-9527 Sigma, cellulose membrane). Superparamagnetic nanoparticles were prepared by alkaline co-precipitation of ferric and ferrous chlorides in aqueous solution as already described elsewhere in detail [12]. For the polymer coating, the iron oxide suspension was mixed at various ratios with 25 kDa polyethylenimine PEI (Aldrich). The samples will be referred to as R ¼ 2, 1, 0.4, 0.2, 0.1 in this article according to their PEI:Fe mass ratio. Transmission electron microscopy (TEM) was performed using a Phillips CM-20 microscope operating at 200 kV. For sample preparation, dilute drops of suspensions were allowed to dry slowly on carbon-coated copper grids. The particle size distribution was estimated by measuring 360 particles at a magnification of 80,000. Size and size distribution were determined at 901 on a photon correlation spectrometer (PCS) from Brookhaven equipped with a BI-9000AT digital autocorrelator. The CONTIN method was used for data processing.
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The concentration of iron oxide nanoparticles was set to 0.015 mg iron/ml for all measurements. The sizes of the SPION–PEI–DNA complexes were measured at a N/P ratio (ratio of nitrogen-containing groups of the polymer to phosphate groups of the nucleic acid) of 7.5, assuming the DNA was entirely complexed. The nitrogen content of PEI has been measured and calculated according to Harpe et al. [13], and the phosphate content was calculated from the size of the plasmid (4.7 kb). The theoretical refractive index of magnetite (2.42 [14]) was used to calculate the numberweighted distribution from the raw-intensity weighted data. x-potential measurements were performed using the same setting, equipped with a platinum electrode. The electrode was cleaned for 10 min in an ultrasonic bath before each measurement and pre-equilibrated for 2 min in an aliquot of the sample. For size and x-potential measurements, the suspensions were diluted in 10 mM phosphate buffer (pH 7.4). Viscosity, refractive index, and dielectric constant of pure water were used to characterize the dispersing medium. The sedimentation behaviour of the nanoparticles in the presence of a magnetic field was investigated photometrically (Perkin-Elmer UV spectrophotometer 9). An NdFeB magnet (Maurer Magnets, Switzerland, Br ¼ 300 mT) was attached to the bottom of a polystyrene UV cuvette that has been adapted in order to perform the measurement at a specific distance (4 mm) from the permanent magnet. The dispersion was diluted in 10 mM phosphate buffer to 100 mg Fe/ml, 0.5 ml were transferred to the cuvette, and the absorption was measured at 500 nm for 100 min. The same experiments were repeated with SPION–PEI–DNA complexes at a concentration of 20 mg Fe/ml. The colloidal stability of four different polymer/iron mass ratios was investigated by turbidity measurements. The dispersions were mixed with differently concentrated sodium chloride and calcium chloride solutions thereby setting the iron concentration to100 mg/ml. Calcium chloride was used as a bivalent electrolyte, as the critical coagulation concentration (CCC) depends strongly on the valency of the counterions. After rapid homogenization, the turbidity (s) was measured at 500 nm as a function of time (t) and the aggregation kinetic constant was calculated from the initial slope of s versus t. The critical coagulation concentration of salt was deduced from the stability factor (W) variation as a function of ionic strength plotted in log–log scale, with W defined by the following ratio of fast (ds/dt)f and slow (ds/dt)s aggregation constants [15]: W ¼ (dA/dt)fast/(dA/dt)slow. Turbidity measurements were also carried out to study the effect of pH on the samples. Furthermore, colloidal stability was investigated as a function of salt concentration (at pH 7, 10 mM HEPES) for different polymer/iron mass ratios R ( ¼ 2, 1, 0.4, 0.2) measuring the aggregation kinetics via turbidity measurements at 500 nm. Ethidium bromide (EtBr) displacement tests were carried out with PEI-coated SPIONs. Therefore, DNA solution
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was mixed with EtBr. The composition of the obtained complex is given by a [EtBr]/[P] ratio of 0.25, where [P] is the molar concentration of DNA phosphate groups. At this ratio, which corresponds to one molecule of intercalated EtBr per two DNA base pairs, the maximum of EtBr fluorescence intensity was observed. The PEI-coated SPIONs were added to the DNA/EtBr at different N/P ratios. The observed fluorescence is described as the maximum fluorescence signal of intercalated EtBr compared to the signal in the absence of any competitor for binding. The background fluorescence in the absence of DNA was taken into account.
3.1. PEI-coated iron oxide nanoparticles When the polymer was added to the nanoparticles, an increase in particle size was observed due to polymer adsorption on the nanoparticles and subsequent bead formation. In general, aggregation of fine particle suspensions is usually achieved by adding high molecular weight polymers. The polymers are of sufficient size to extend beyond the electric double layer and they can bridge two or more particles together to form large aggregates [16]. It is now crucial to choose the molecular weight of the used polymer carefully, in order to induce bead formation without causing extensive bridging flocculation. The iron oxide particles were clearly detected by TEM in the polymer matrix. Each nanoparticle associated with more than one strand of the polyethylenimine and, likewise, each strand of polyethylenimine attached to more than one nanoparticle, resulting in a bridging aggregation. This phenomenon was observed for all PEI:Fe mass ratios. An average bead size of 27712 nm was obtained exemplarily for sample R ¼ 2 (PEI:Fe mass ratio of 2) measuring 360 beads by TEM. The PEI iron oxide nanobeads described elsewhere showed an average size of 200 nm, which is attributed to the higher molecular weight (800 kDa) PEI [7]. Also the number of particles is not sufficient for statistical analysis of the particle size distribution, the obtained result correlates very well with the data obtained by dynamic light scattering. All suspensions were investigated by means of photon correlation spectroscopy. Fig. 1 shows the cumulative number weighted size distribution of PEI-coated SPIONs in 10 mM phosphate buffer for different PEI:Fe mass ratios R. A representative bright field transmission electron micrograph showing one SPION–PEI bead is shown in the inlet. The d50 diameter of the formed beads decreased with increasing PEI:Fe ratio indicating that the amount of SPIONs per bead decreased at the same time. Photometrical investigations of the sedimentation behaviour on a permanent magnet confirmed this hypothesis. Uncoated SPIONs sedimented only gradually and larger beads sedimented faster than smaller beads since they
Fig. 1. Cumulative number weighted size distribution of PEI-coated SPIONs measured by PCS in 10 mM phosphate buffer for different PEI:Fe mass ratios R. Inlet: bright field transmission electron micrograph showing one PEI:SPION bead.
15 ζ-Potential [mV]
3. Results and discussion
10 5 0 -5 -10 -15 0
1 2 PEI:Fe mass ratio R
Fig. 2. z-Potential of PEI-coated SPIONs measured in 10 mM phosphate buffer for different PEI:Fe mass ratios R. The arrow indicates the minimal PEI:Fe concentration at which no flocculation was observed after 30 min.
contained more individual SPIONs and therefore more magnetic material. The z-potential of PEI-coated iron oxide beads was measured for different polymer/iron mass ratios R (0.1pRp2) in 10 mM phosphate buffer (pH ¼ 7.4) at an iron concentration of 0.015 mg iron/ml. The shape of the z-potential was characteristic of an adsorption phenomenon, and two distinct areas could be observed (Fig. 2). The z-potential increased with increasing PEI:Fe ratio rapidly to a critical value of Rc ¼ 0.2. Here, the negative surface charge of the SPIONs was shielded. For R4Rc, the z-potential remained constant. For PEI:Fe ratios 0.2oRo0.4 the dispersion was turbid, but no flocculation was observed. However, flocculation started at PEI:Fe ratios Ro0.2 in 10 mM phosphate buffer, due to insufficient stabilization of the SPIONs by the polymer. Higher polyelectrolyte concentrations changed the charge of the particles surface. These results suggested that at RoRc, the surface of the particles was not completely covered by the adsorbed polymer and Coulomb interactions still existed between the fully and partially covered SPIONs. At higher polymer concentration (R4Rc), the iron oxide core was completely embedded in the beads and the polymer electrostatically and sterically stabilized the beads.
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(dA/dt)pH R = 0 (dA/dt)pH R = 2
dA/dt
0.10
0.05
0.00 2
4
6
8 pH
10
12
14
Fig. 3. Initial slope of the turbidity as a function of pH for uncoated SPIONs (R ¼ 0) and PEI-coated SPIONs (R ¼ 2).
2.0
R = 2.0 R = 1.1 R = 0.4 R = 0.2
1.5 log W
Coulomb forces as well as van der Waals interactions were responsible for the adsorption of PEI to the particles’ surface. The configuration of the adsorbed polymer is difficult to determine, and is affected by the polymer concentration and molecular weight, pH, ionic strength, or surface charge. To study the coating mechanism, it is very helpful to work with particles that are uniform in size and shape. A number of such model systems have been studied in the last years using inorganic particles as a core and various polymers as shells. The colloidal stability of uncoated iron oxide nanoparticles and PEI-coated particles was investigated at a PEI:Fe ratio R ¼ 2 by studying the change of turbidity (dA/dt) at a given electrolyte concentration (2.5 mM NaCl) at various pH. The obtained results are shown in Fig. 3. Electrostatic forces purely stabilized uncoated iron oxide nanoparticles. The nanoparticles exhibited good colloidal stability below pH of 5 and above pH 11. In the pH range from 5 to 11, agglomeration of the particles was observed. This behaviour was attributed to the surface potential of the nanoparticles: At pHo5 and pH411, the particles possessed enough charges on the surface to prevent aggregation, whereas, at pH close to the IEP ¼ 7 [14], the electrostatic repulsion between the particles was not sufficiently high for stabilizing the system and therefore flocculation occurred. No change in turbidity was observed for PEI stabilized nanoparticles between pH 3 and 12. In a second study, the colloidal stability of the PEI beads was investigated at different electrolyte concentrations at constant pH (Fig. 4). The differences in flocculation provided a simple means to detect modification in the surface characteristics of iron oxide beads and the stability of polymer-coated particles was closely related to the conformation of the adsorbed polymer [17]. The CCC was extracted at the intercept point between fast and slow coagulation. The polymer-coated nanoparticles were remarkably stable even in sodium chloride solution (5 M), where no change in turbidity and hence no agglomeration could be detected (data not shown). Therefore, it was impossible to obtain data corresponding to a fast coagulation process, and the
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1.0 0.5 0.0 1.5
2.0 2.5 3.0 log c [mM CaCl2]
3.5
Fig. 4. –A log plot of the stability factor (W) variation as function of ionic strength for different PEI-coated SPIONs (R ¼ 2, 1.1, 0.4, 0.2).
CCC was higher than 5 M NaCl. In order to compare the stabilization of the different beads, CaCl2 was used as a bivalent electrolyte. Linear changes in the early stages of the aggregation process were observed upon salt addition. The aggregation rate was proportional to the number of particles, and the initial linear regime of particle aggregation was more pronounced for R ¼ 0.2. Aggregation was only induced at high electrolyte concentration as all primary amino-groups of the polymer are charged at physiological pH [13], thus ensuring electrostatic stabilization of the beads. The aggregation behaviour was comparable for most PEI:Fe mass ratios and a CCC of 2.1 (R ¼ 0.2) to 3.1 (R ¼ 1) was determined. The CCC was shifted considerably for R ¼ 0.2, when the surface of the SPIONs was insufficiently covered by the polymer. The high CCC values indicated a long-term colloidal stability of the beads. No increase in particle sizes and hence no agglomeration was measured for nanoparticles over a time period of 3 months. 3.2. Particle interaction with plasmid DNA The surface charge of gene delivery systems is known to be one of the major factors influencing their biodistribution [18] and transfection efficiency [19]. In the common literature, it was shown that a positive net charge and hence a positive z-potential was necessary for complexes entering the cell [19] and that the uptake of 50 nm particles compared to 200 nm particles was up to 10 times faster [15], indicating that small particles facilitated endocytosis [20]. In a recent study, we confirmed that cellular uptake [21,22] is influenced by the surface charge and the size of the SPION–PEI–DNA complexes. The behaviour of the z-potential of PEI-coated beads with changing amounts of adsorbed DNA is shown in Fig. 5. The results are given in N/P ratios, which refer to the ratio of polymer nitrogen to DNA phosphates and describe the amount of polymer needed for complex formation. The positive charge of the polymer-coated SPIONs (high N/P ratio) was diminished upon DNA addition and a negative surface potential was measured for higher concentrations
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304
R = 2.0, IEP(N/P) = 3.5 R = 1.1, IEP(N/P) = 4.6 R = 0.4, IEP(N/P) = 5.8 ζ-Potential [mV]
20
0 1 -20
10 N/P
Fig. 5. Variation of the z-potential as a function of the N/P ratio and the PEI:Fe mass ratio R.
of additional DNA (low N/P). The isoelectric point decreased with increasing PEI:Fe mass ratio R corresponding to smaller particles (see Fig. 2). Smaller particles provide a larger surface per mass requiring therefore more DNA to compensate the higher positive charge. The ability of PEI-coated SPIONs to effectively bind DNA was monitored by fluorescence measurements of DNA–EtBr complexes. The fluorescence intensity decreased progressively with increasing N/P ratio up to N/P1. For N/P41, the fluorescence remained more or less constant at 20–30% of its original fluorescence. This significant fluorescence quenching indicates that the formation of the SPION– PEI–DNA complexes is not in favor of the interaction between the DNA and EtBr. It is known that high density of negatively charged phosphate groups of the double helix provides the ability of DNA to form rather stable complexes with synthetic polycations [7]. Studies showed that the decrease in fluorescence could be explained by the complexation of DNA with the polymer resulting in a conformational change in the secondary structure of DNA from the B- to C-form [23]. Although both size and isoelectric point changed for the investigated systems, the results showed clearly that the complexation of DNA depended strongly on the polymer concentration rather than the amount of SPIONs; the size of the complexes and their different PEI:Fe ratios did not influence the ability of PEI to form complexes with DNA. In a further study, the colloidal stability of SPION– PEI–DNA complexes (R ¼ 2, N/P ¼ 7.5) was tested in different cell media such as DMEM LG, DMEM HG, DMEM LG+10% fetal calf serum (FCS) and DMEM HG+10% FCS. The beads agglomerated immediately in the presence of FCS, whereas they remained stable in all media without FCS and no change in the turbidity as well as in the bead size could be observed for 1 h. This result was consistent with previous reports that PEI interacts unspecifically with negatively charged molecules in the serum as well as plasma proteins such as opsonins [9]. In the corresponding sedimentation experiment, SPION–PEI–DNA complexes settled significantly faster (95% within 5 min) on a permanent magnet than the
original beads without DNA (11% within 5 min). The complex formation between negatively charged DNA and positively charged PEI-coated SPIONs is driven by electrostatic attraction. The formation of the SPION– PEI–DNA complexes was very likely to take place via bead agglomeration, although the hydrodynamic diameter increased only slightly from 3879 nm, without DNA, to 52717 nm, with DNA. Several single SPION–PEI beads were associated entrapping the plasmid DNA with a conserved positive net potential. The total and fast sedimentation of all SPION beads associated with DNA proved that all SPIONs must be embedded within the newly formed complexes. 3.3. SPION–PEI–DNA complexes for non-viral gene delivery In order to maximize the transfection efficiencies, the DNA entry into the cells and the nuclear uptake of the DNA for its expression has to be enhanced. We have just reported [22] how gene delivery can be improved using permanent and pulsating magnetic fields. The transfection efficiency was evaluated for different N/P ratios of the smallest particles synthesized (R ¼ 2), as these particles showed best transfection efficiency after initial screening. Efficient transfection was only observed for N/P ratios with a positive z-potential and transfection rates of 34.5% were achieved at N/P ratios of 7.6, which were accompanied by low cytotoxicity. Interestingly, SPION–PEI–DNA complexes showed less toxicity than PEI–DNA complexes alone. Concentrations of PEI-DNA complexes of up to 10 mg/ml PEI with an N/P ratio of 2–10 showed 1073.2% dead COS cells. In comparison, comparable SPION– PEI–DNA showed just 2.870.32% of dead cells, confirmed by PI staining and FACS analysis. Therefore, it can be deduced that the screening of the charge upon complexation of the polymer reduces the toxicity of this transfection system. Furthermore, the influence of particle size on the transfection efficiency of the SPION–PEI–DNA complex was evaluated. Therefore, different sizes of SPION–PEI–DNA complexes were tested as transfection agents. With the large particles (200 nm), only very low transfection rates of up to 0.6% could be achieved with COS cells, whereas smaller particles (around 60 nm) resulted in much higher transfection rates (34.5%). In our study, the applied magnetic field reduced the free diffusion of the particles, which could have resulted in the higher uptake of nanoparticles. 4. Summary This work describes the formulation of different PEIcoated superparamagnetic iron oxide nanoparticles for gene transfection. We applied colloidal and interface science principles for the development of an effective transfection system, which proved to be highly sufficient. Different sizes of SPION–PEI beads can be produced and
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used as complexation agents for DNA by electrostatic interaction. Acknowledgements This project was funded by the Vetsuisse-Faculty of the Universities of Berne and Zurich, Switzerland. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
A.K. Gupta, M. Gupta, Biomaterials 26/18 (2005) 3995. A.K. Gupta, A.S.G. Curtis, Biomaterials 25/15 (2004) 3029. Y. Zhang, N. Kohler, M. Zhang, Biomaterials 23/7 (2002) 1553. L.E. Euliss, S.G. Grancharov, S. O’Brien, et al., Nano Lett. 3/11 (2003) 1489. S. Mehier-Humbert, R.H. Guy, Adv. Drug Deliv. Rev. 57 (2005) 733. C. Plank, M. Anton, C. Rudolph, et al., Expert Opin. Biol. Ther. 3/5 (2003) 745. S. Huth, J. Lausier, S.W. Gersting, et al., J. Gene Med. 6/8 (2004) 923. J. Rejman, V. Oberle, I.S. Zuhorn, et al., J. Biochem. 377/1 (2004) 159. M. Neu, D. Fischer, T. Kissel, J. Gene Med. 7/8 (2005) 992. H. Yin, H.P. Too, G.M. Chow, Biomaterials 26/29 (2005) 5818.
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[11] U. Lungwitz, M. Breunig, T. Blunk, et al., Eur. J. Pharm. Biopharm. 60/2 (2005) 247. [12] M. Chastellain, A. Petri, H. Hofmann, J. Colloid Interface Sci. 278/2 (2004) 353. [13] A. von Harpe, H. Petersen, Y. Li, et al., J. Control Release 69/2 (2000) 309. [14] R.M. Cornell, U. Schwertmann, The Iron Oxides, VCH Verlagsgesellshaft mbH, Weinheim, 1996. [15] R.M. Santos, J. Forcada, J. Polym. Sci. Part A: Polym. Chem. 37 (1999) 501. [16] P.D. Yates, G.V. Franks, S. Briggs, et al., Colloids Surf. A: Physiochem. Eng. Aspects 255 (2005) 85. [17] D.H. Napper, A. Netschey, J. Colloid Interface Sci. 37/3 (1971) 528–535. [18] R.I. Mahato, Y. Takakura, M. Hashida, Crit. Rev. Ther. Drug Carr. Syst. 14/2 (1997) 133. [19] T. Nomura, S. Nakjima, K. Kawabata, et al., Cancer Res. 57/13 (1997) 2681. [20] K. Kunath, A. von Harpe, D. Fischer, et al., J. Control. Release 88/1 (2003) 159. [21] A. Fink-Petri, M. Chastellain, L. Juillerat-Jeanneret, et al., Biomaterials 26 (2005) 2685. [22] S.W. Kamau, P.O. Hassa, B. Steitz, et al., Nucleic Acids Res. 34/5 (2006) e40. [23] Y. Zhou, Y. Li, Biophys. Chem. 107 (2004) 273.
Journal of Magnetism and Magnetic Materials 311 (2007) 300–305 www.elsevier.com/locate/jmmm
Characterization of PEI-coated superparamagnetic iron oxide nanoparticles for transfection: Size distribution, colloidal properties and DNA interaction Benedikt Steitza, Heinrich Hofmanna, Sarah W. Kamaub, Paul O. Hassab, Michael O. Hottigerb, Brigitte von Rechenbergc, Magarethe Hofmann-Amtenbrinkd, Alke Petri-Finka, a
Laboratory of Powder Technology, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Lausanne, Switzerland b Institute of Veterinary Biochemistry and Molecular Biology, University of Zu¨rich, Zu¨rich, Switzerland c Musculoskeletal Research Unit, Equine Hospital, Vetsuisse Faculty Zurich, University of Zurich, Winterthurerstr. 260, 8057 Zurich, Switzerland d MatSearch, Chemin Jean Pavillard 14, 1009 Pully, Switzerland Available online 20 December 2006
Abstract Superparamagnetic iron oxide nanoparticles (SPIONs) were coated with polyethylenimine. Here, we briefly describe the synthesis as well as DNA:PEI:SPION complexes and the characterization of the compounds according to their particle size, z-potential, morphology, DNA complexing ability, magnetic sedimentation, and colloidal stability. PEI coating of SPIONs led to colloidally stable beads even in high salt concentrations over a wide pH range. DNA plasmids and PCR products encoding for green fluorescent protein were associated with the described beads. The complexes were added to cells and exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency. r 2006 Elsevier B.V. All rights reserved. Keywords: Superparamagnetism; Iron oxide; Transfection; Gene delivery; Colloidal stability
1. Introduction The possible applications of superparamagnetic iron oxide nanoparticles (SPIONs) are manifold ranging from various separation techniques and MRI contrast agents to drug delivery systems, magnetic hyperthermia, and magnetically assisted transfection [1]. Different coatings such as phospholipids, fatty acids, surfactants, silica, polymers, polysaccharides, and peptides of superparamagnetic nanoparticles are reported in the literature [2–4]. Various gene delivery vectors have been investigated which can be divided into two main groups: biological vectors where the gene of interest is cloned into the virus genome, and techniques employing either chemical or physical approaches such as cationic molecular carriers Corresponding author. Tel.: +41 21 693 5107; fax: +41 21 693 3089.
E-mail address: alke.fink@epfl.ch (A. Petri-Fink). 0304-8853/$ - see front matter r 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2006.10.1194
that form electrostatic interactions with DNA [5]. Magnetically assisted transfection now combines superparamagnetic nanoparticles with DNA. Plank et al. [6] have studied this method extensively. A force parallel to the field gradient acts on the magnetized particles and due to this magnetic field gradient the particles sediment on the cell surface [7]. The potential of magnetically assisted transfection might be seen in the efficient and fast delivery of genetic materials into the cells nucleus and the enhancement of gene transfection in vivo and in vitro with minimal toxicity to the cells. One important factor influencing the behaviour of the transfection agent is the size of the complexes. The size of nanoparticles has been shown to influence the rate of their uptake as well as their cytoxicity. Commercial products such as transMAGPEI (Chemicell, Germany) are relatively large particles with a size of 200 nm [7]. Rejman et al. [8] showed that nanoparticles with a size of 50 nm are
ARTICLE IN PRESS B. Steitz et al. / Journal of Magnetism and Magnetic Materials 311 (2007) 300–305
taken up 3–4 times faster than 100 nm particles and 8–10 times faster than 200 and 500 nm particles. Endocytosis is not only more efficient with 50 nm particles, but the velocity of cytoplasmatic movement was also found to be a function of particle size [9]. Estimated from the Stoke– Einstein relation, the diffusion coefficient of 50 nm nanoparticles compared to 200 nm particles is 4 times larger. In a previous study, the influence of the size of nickel ferrite nanoparticles on the cytotoxicity was determined [10]. In this study, 10 nm nanoparticles were compared to 150 nm particles, the latter showing a higher cytotoxicity than the 10 nm particles. In the present study, the characteristics of SPIONs coated with polyethylenimine (PEI) are investigated and first results about the applicability as transfection agents are reported. PEI is known to form cationic complexes, which interact non-specifically with negatively charged species such as DNA and enter the cell via endocytosis [11]. PEI offers a high positive charge density and exhibits a strong proton buffer capacity over a broad pH range. The latter characteristic is advantageous for PEI since the proton sponge effect allows sufficient gene escape without the necessity of adding endosome disrupting reagents, such as chloroquine, poly(propylacrylic acid), or peptides. Here, we briefly describe the synthesis of polymer coated SPIONs as well as SPION–PEI–DNA complexes and the characterization of the compounds with respect to their particle size, x-potential, morphology, DNA complexing ability, magnetic sedimentation, and colloidal stability, all with regard to their applicability as non-viral gene vectors. 2. Materials and methods All chemicals were of analytical reagent grade and used without further purification. Ultra-pure deionized water (Seralpur delta UV/UF setting, 0.055 mS/cm) was used in all synthesis steps. Dialysis tubing with a molecular weight cut-off at 12,000 was used for dialysis (D-9527 Sigma, cellulose membrane). Superparamagnetic nanoparticles were prepared by alkaline co-precipitation of ferric and ferrous chlorides in aqueous solution as already described elsewhere in detail [12]. For the polymer coating, the iron oxide suspension was mixed at various ratios with 25 kDa polyethylenimine PEI (Aldrich). The samples will be referred to as R ¼ 2, 1, 0.4, 0.2, 0.1 in this article according to their PEI:Fe mass ratio. Transmission electron microscopy (TEM) was performed using a Phillips CM-20 microscope operating at 200 kV. For sample preparation, dilute drops of suspensions were allowed to dry slowly on carbon-coated copper grids. The particle size distribution was estimated by measuring 360 particles at a magnification of 80,000. Size and size distribution were determined at 901 on a photon correlation spectrometer (PCS) from Brookhaven equipped with a BI-9000AT digital autocorrelator. The CONTIN method was used for data processing.
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The concentration of iron oxide nanoparticles was set to 0.015 mg iron/ml for all measurements. The sizes of the SPION–PEI–DNA complexes were measured at a N/P ratio (ratio of nitrogen-containing groups of the polymer to phosphate groups of the nucleic acid) of 7.5, assuming the DNA was entirely complexed. The nitrogen content of PEI has been measured and calculated according to Harpe et al. [13], and the phosphate content was calculated from the size of the plasmid (4.7 kb). The theoretical refractive index of magnetite (2.42 [14]) was used to calculate the numberweighted distribution from the raw-intensity weighted data. x-potential measurements were performed using the same setting, equipped with a platinum electrode. The electrode was cleaned for 10 min in an ultrasonic bath before each measurement and pre-equilibrated for 2 min in an aliquot of the sample. For size and x-potential measurements, the suspensions were diluted in 10 mM phosphate buffer (pH 7.4). Viscosity, refractive index, and dielectric constant of pure water were used to characterize the dispersing medium. The sedimentation behaviour of the nanoparticles in the presence of a magnetic field was investigated photometrically (Perkin-Elmer UV spectrophotometer 9). An NdFeB magnet (Maurer Magnets, Switzerland, Br ¼ 300 mT) was attached to the bottom of a polystyrene UV cuvette that has been adapted in order to perform the measurement at a specific distance (4 mm) from the permanent magnet. The dispersion was diluted in 10 mM phosphate buffer to 100 mg Fe/ml, 0.5 ml were transferred to the cuvette, and the absorption was measured at 500 nm for 100 min. The same experiments were repeated with SPION–PEI–DNA complexes at a concentration of 20 mg Fe/ml. The colloidal stability of four different polymer/iron mass ratios was investigated by turbidity measurements. The dispersions were mixed with differently concentrated sodium chloride and calcium chloride solutions thereby setting the iron concentration to100 mg/ml. Calcium chloride was used as a bivalent electrolyte, as the critical coagulation concentration (CCC) depends strongly on the valency of the counterions. After rapid homogenization, the turbidity (s) was measured at 500 nm as a function of time (t) and the aggregation kinetic constant was calculated from the initial slope of s versus t. The critical coagulation concentration of salt was deduced from the stability factor (W) variation as a function of ionic strength plotted in log–log scale, with W defined by the following ratio of fast (ds/dt)f and slow (ds/dt)s aggregation constants [15]: W ¼ (dA/dt)fast/(dA/dt)slow. Turbidity measurements were also carried out to study the effect of pH on the samples. Furthermore, colloidal stability was investigated as a function of salt concentration (at pH 7, 10 mM HEPES) for different polymer/iron mass ratios R ( ¼ 2, 1, 0.4, 0.2) measuring the aggregation kinetics via turbidity measurements at 500 nm. Ethidium bromide (EtBr) displacement tests were carried out with PEI-coated SPIONs. Therefore, DNA solution
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was mixed with EtBr. The composition of the obtained complex is given by a [EtBr]/[P] ratio of 0.25, where [P] is the molar concentration of DNA phosphate groups. At this ratio, which corresponds to one molecule of intercalated EtBr per two DNA base pairs, the maximum of EtBr fluorescence intensity was observed. The PEI-coated SPIONs were added to the DNA/EtBr at different N/P ratios. The observed fluorescence is described as the maximum fluorescence signal of intercalated EtBr compared to the signal in the absence of any competitor for binding. The background fluorescence in the absence of DNA was taken into account.
3.1. PEI-coated iron oxide nanoparticles When the polymer was added to the nanoparticles, an increase in particle size was observed due to polymer adsorption on the nanoparticles and subsequent bead formation. In general, aggregation of fine particle suspensions is usually achieved by adding high molecular weight polymers. The polymers are of sufficient size to extend beyond the electric double layer and they can bridge two or more particles together to form large aggregates [16]. It is now crucial to choose the molecular weight of the used polymer carefully, in order to induce bead formation without causing extensive bridging flocculation. The iron oxide particles were clearly detected by TEM in the polymer matrix. Each nanoparticle associated with more than one strand of the polyethylenimine and, likewise, each strand of polyethylenimine attached to more than one nanoparticle, resulting in a bridging aggregation. This phenomenon was observed for all PEI:Fe mass ratios. An average bead size of 27712 nm was obtained exemplarily for sample R ¼ 2 (PEI:Fe mass ratio of 2) measuring 360 beads by TEM. The PEI iron oxide nanobeads described elsewhere showed an average size of 200 nm, which is attributed to the higher molecular weight (800 kDa) PEI [7]. Also the number of particles is not sufficient for statistical analysis of the particle size distribution, the obtained result correlates very well with the data obtained by dynamic light scattering. All suspensions were investigated by means of photon correlation spectroscopy. Fig. 1 shows the cumulative number weighted size distribution of PEI-coated SPIONs in 10 mM phosphate buffer for different PEI:Fe mass ratios R. A representative bright field transmission electron micrograph showing one SPION–PEI bead is shown in the inlet. The d50 diameter of the formed beads decreased with increasing PEI:Fe ratio indicating that the amount of SPIONs per bead decreased at the same time. Photometrical investigations of the sedimentation behaviour on a permanent magnet confirmed this hypothesis. Uncoated SPIONs sedimented only gradually and larger beads sedimented faster than smaller beads since they
Fig. 1. Cumulative number weighted size distribution of PEI-coated SPIONs measured by PCS in 10 mM phosphate buffer for different PEI:Fe mass ratios R. Inlet: bright field transmission electron micrograph showing one PEI:SPION bead.
15 ζ-Potential [mV]
3. Results and discussion
10 5 0 -5 -10 -15 0
1 2 PEI:Fe mass ratio R
Fig. 2. z-Potential of PEI-coated SPIONs measured in 10 mM phosphate buffer for different PEI:Fe mass ratios R. The arrow indicates the minimal PEI:Fe concentration at which no flocculation was observed after 30 min.
contained more individual SPIONs and therefore more magnetic material. The z-potential of PEI-coated iron oxide beads was measured for different polymer/iron mass ratios R (0.1pRp2) in 10 mM phosphate buffer (pH ¼ 7.4) at an iron concentration of 0.015 mg iron/ml. The shape of the z-potential was characteristic of an adsorption phenomenon, and two distinct areas could be observed (Fig. 2). The z-potential increased with increasing PEI:Fe ratio rapidly to a critical value of Rc ¼ 0.2. Here, the negative surface charge of the SPIONs was shielded. For R4Rc, the z-potential remained constant. For PEI:Fe ratios 0.2oRo0.4 the dispersion was turbid, but no flocculation was observed. However, flocculation started at PEI:Fe ratios Ro0.2 in 10 mM phosphate buffer, due to insufficient stabilization of the SPIONs by the polymer. Higher polyelectrolyte concentrations changed the charge of the particles surface. These results suggested that at RoRc, the surface of the particles was not completely covered by the adsorbed polymer and Coulomb interactions still existed between the fully and partially covered SPIONs. At higher polymer concentration (R4Rc), the iron oxide core was completely embedded in the beads and the polymer electrostatically and sterically stabilized the beads.
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(dA/dt)pH R = 0 (dA/dt)pH R = 2
dA/dt
0.10
0.05
0.00 2
4
6
8 pH
10
12
14
Fig. 3. Initial slope of the turbidity as a function of pH for uncoated SPIONs (R ¼ 0) and PEI-coated SPIONs (R ¼ 2).
2.0
R = 2.0 R = 1.1 R = 0.4 R = 0.2
1.5 log W
Coulomb forces as well as van der Waals interactions were responsible for the adsorption of PEI to the particles’ surface. The configuration of the adsorbed polymer is difficult to determine, and is affected by the polymer concentration and molecular weight, pH, ionic strength, or surface charge. To study the coating mechanism, it is very helpful to work with particles that are uniform in size and shape. A number of such model systems have been studied in the last years using inorganic particles as a core and various polymers as shells. The colloidal stability of uncoated iron oxide nanoparticles and PEI-coated particles was investigated at a PEI:Fe ratio R ¼ 2 by studying the change of turbidity (dA/dt) at a given electrolyte concentration (2.5 mM NaCl) at various pH. The obtained results are shown in Fig. 3. Electrostatic forces purely stabilized uncoated iron oxide nanoparticles. The nanoparticles exhibited good colloidal stability below pH of 5 and above pH 11. In the pH range from 5 to 11, agglomeration of the particles was observed. This behaviour was attributed to the surface potential of the nanoparticles: At pHo5 and pH411, the particles possessed enough charges on the surface to prevent aggregation, whereas, at pH close to the IEP ¼ 7 [14], the electrostatic repulsion between the particles was not sufficiently high for stabilizing the system and therefore flocculation occurred. No change in turbidity was observed for PEI stabilized nanoparticles between pH 3 and 12. In a second study, the colloidal stability of the PEI beads was investigated at different electrolyte concentrations at constant pH (Fig. 4). The differences in flocculation provided a simple means to detect modification in the surface characteristics of iron oxide beads and the stability of polymer-coated particles was closely related to the conformation of the adsorbed polymer [17]. The CCC was extracted at the intercept point between fast and slow coagulation. The polymer-coated nanoparticles were remarkably stable even in sodium chloride solution (5 M), where no change in turbidity and hence no agglomeration could be detected (data not shown). Therefore, it was impossible to obtain data corresponding to a fast coagulation process, and the
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1.0 0.5 0.0 1.5
2.0 2.5 3.0 log c [mM CaCl2]
3.5
Fig. 4. –A log plot of the stability factor (W) variation as function of ionic strength for different PEI-coated SPIONs (R ¼ 2, 1.1, 0.4, 0.2).
CCC was higher than 5 M NaCl. In order to compare the stabilization of the different beads, CaCl2 was used as a bivalent electrolyte. Linear changes in the early stages of the aggregation process were observed upon salt addition. The aggregation rate was proportional to the number of particles, and the initial linear regime of particle aggregation was more pronounced for R ¼ 0.2. Aggregation was only induced at high electrolyte concentration as all primary amino-groups of the polymer are charged at physiological pH [13], thus ensuring electrostatic stabilization of the beads. The aggregation behaviour was comparable for most PEI:Fe mass ratios and a CCC of 2.1 (R ¼ 0.2) to 3.1 (R ¼ 1) was determined. The CCC was shifted considerably for R ¼ 0.2, when the surface of the SPIONs was insufficiently covered by the polymer. The high CCC values indicated a long-term colloidal stability of the beads. No increase in particle sizes and hence no agglomeration was measured for nanoparticles over a time period of 3 months. 3.2. Particle interaction with plasmid DNA The surface charge of gene delivery systems is known to be one of the major factors influencing their biodistribution [18] and transfection efficiency [19]. In the common literature, it was shown that a positive net charge and hence a positive z-potential was necessary for complexes entering the cell [19] and that the uptake of 50 nm particles compared to 200 nm particles was up to 10 times faster [15], indicating that small particles facilitated endocytosis [20]. In a recent study, we confirmed that cellular uptake [21,22] is influenced by the surface charge and the size of the SPION–PEI–DNA complexes. The behaviour of the z-potential of PEI-coated beads with changing amounts of adsorbed DNA is shown in Fig. 5. The results are given in N/P ratios, which refer to the ratio of polymer nitrogen to DNA phosphates and describe the amount of polymer needed for complex formation. The positive charge of the polymer-coated SPIONs (high N/P ratio) was diminished upon DNA addition and a negative surface potential was measured for higher concentrations
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R = 2.0, IEP(N/P) = 3.5 R = 1.1, IEP(N/P) = 4.6 R = 0.4, IEP(N/P) = 5.8 ζ-Potential [mV]
20
0 1 -20
10 N/P
Fig. 5. Variation of the z-potential as a function of the N/P ratio and the PEI:Fe mass ratio R.
of additional DNA (low N/P). The isoelectric point decreased with increasing PEI:Fe mass ratio R corresponding to smaller particles (see Fig. 2). Smaller particles provide a larger surface per mass requiring therefore more DNA to compensate the higher positive charge. The ability of PEI-coated SPIONs to effectively bind DNA was monitored by fluorescence measurements of DNA–EtBr complexes. The fluorescence intensity decreased progressively with increasing N/P ratio up to N/P1. For N/P41, the fluorescence remained more or less constant at 20–30% of its original fluorescence. This significant fluorescence quenching indicates that the formation of the SPION– PEI–DNA complexes is not in favor of the interaction between the DNA and EtBr. It is known that high density of negatively charged phosphate groups of the double helix provides the ability of DNA to form rather stable complexes with synthetic polycations [7]. Studies showed that the decrease in fluorescence could be explained by the complexation of DNA with the polymer resulting in a conformational change in the secondary structure of DNA from the B- to C-form [23]. Although both size and isoelectric point changed for the investigated systems, the results showed clearly that the complexation of DNA depended strongly on the polymer concentration rather than the amount of SPIONs; the size of the complexes and their different PEI:Fe ratios did not influence the ability of PEI to form complexes with DNA. In a further study, the colloidal stability of SPION– PEI–DNA complexes (R ¼ 2, N/P ¼ 7.5) was tested in different cell media such as DMEM LG, DMEM HG, DMEM LG+10% fetal calf serum (FCS) and DMEM HG+10% FCS. The beads agglomerated immediately in the presence of FCS, whereas they remained stable in all media without FCS and no change in the turbidity as well as in the bead size could be observed for 1 h. This result was consistent with previous reports that PEI interacts unspecifically with negatively charged molecules in the serum as well as plasma proteins such as opsonins [9]. In the corresponding sedimentation experiment, SPION–PEI–DNA complexes settled significantly faster (95% within 5 min) on a permanent magnet than the
original beads without DNA (11% within 5 min). The complex formation between negatively charged DNA and positively charged PEI-coated SPIONs is driven by electrostatic attraction. The formation of the SPION– PEI–DNA complexes was very likely to take place via bead agglomeration, although the hydrodynamic diameter increased only slightly from 3879 nm, without DNA, to 52717 nm, with DNA. Several single SPION–PEI beads were associated entrapping the plasmid DNA with a conserved positive net potential. The total and fast sedimentation of all SPION beads associated with DNA proved that all SPIONs must be embedded within the newly formed complexes. 3.3. SPION–PEI–DNA complexes for non-viral gene delivery In order to maximize the transfection efficiencies, the DNA entry into the cells and the nuclear uptake of the DNA for its expression has to be enhanced. We have just reported [22] how gene delivery can be improved using permanent and pulsating magnetic fields. The transfection efficiency was evaluated for different N/P ratios of the smallest particles synthesized (R ¼ 2), as these particles showed best transfection efficiency after initial screening. Efficient transfection was only observed for N/P ratios with a positive z-potential and transfection rates of 34.5% were achieved at N/P ratios of 7.6, which were accompanied by low cytotoxicity. Interestingly, SPION–PEI–DNA complexes showed less toxicity than PEI–DNA complexes alone. Concentrations of PEI-DNA complexes of up to 10 mg/ml PEI with an N/P ratio of 2–10 showed 1073.2% dead COS cells. In comparison, comparable SPION– PEI–DNA showed just 2.870.32% of dead cells, confirmed by PI staining and FACS analysis. Therefore, it can be deduced that the screening of the charge upon complexation of the polymer reduces the toxicity of this transfection system. Furthermore, the influence of particle size on the transfection efficiency of the SPION–PEI–DNA complex was evaluated. Therefore, different sizes of SPION–PEI–DNA complexes were tested as transfection agents. With the large particles (200 nm), only very low transfection rates of up to 0.6% could be achieved with COS cells, whereas smaller particles (around 60 nm) resulted in much higher transfection rates (34.5%). In our study, the applied magnetic field reduced the free diffusion of the particles, which could have resulted in the higher uptake of nanoparticles. 4. Summary This work describes the formulation of different PEIcoated superparamagnetic iron oxide nanoparticles for gene transfection. We applied colloidal and interface science principles for the development of an effective transfection system, which proved to be highly sufficient. Different sizes of SPION–PEI beads can be produced and
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used as complexation agents for DNA by electrostatic interaction. Acknowledgements This project was funded by the Vetsuisse-Faculty of the Universities of Berne and Zurich, Switzerland. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
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