Oligonucleotide-protamine-albumin nanoparticles: Protamine sulfate causes drastic size reduction

Journal of Controlled Release 106 (2005) 181 – 187 www.elsevier.com/locate/jconrel

Oligonucleotide-protamine-albumin nanoparticles: Protamine sulfate causes drastic size reduction Gottfried Mayer a, Vitali Vogel a, Jo¨rg Weyermann b, Dirk Lochmann b, Jacomina A. van den Broek a, Christos Tziatzios a, Winfried Haase c, Daan Wouters d, Ulrich S. Schubert d, Andreas Zimmer e, Jo¨rg Kreuter b,*, Dieter Schubert a a

Institut fu¨r Biophysik, Johann Wolfgang Goethe-Universita¨t, Theodor-Stern-Kai 7, Haus 74, 60590 Frankfurt am Main, Germany Institut fu¨r Pharmazeutische Technologie, Johann Wolfgang Goethe-Universita¨t, Marie-Curie-Strasse 9, 60439 Frankfurt am Main, Germany c Max-Planck-Institut fu¨r Biophysik, Marie-Curie-Strasse 15, 60439 Frankfurt am Main, Germany d Laboratory of Macromolecular Chemistry and Nanoscience, Eindhoven University of Technology, NL-5600 MB Eindhoven, The Netherlands e Institut fu¨r Pharmazeutische Chemie und Pharmazeutische Technologie, Karl-Franzens-Universita¨t, Schubertstrasse 6, 8010 Graz, Austria b

Received 2 December 2004; accepted 20 April 2005 Available online 6 July 2005

Abstract Nanoparticles prepared by self-assembly from oligonucleotides (ONs), protamine free base, and human serum albumin (bternary proticlesQ) are spheres of diameters around 200 nm. Substitution of the protamine free base by protamine sulfate leads to proticles of only around 40 nm in diameter with otherwise unchanged properties. The availability of drug delivery systems of very similar composition but grossly different size may be advantageous when dealing with cells which show size-dependent particle uptake. These nanoparticles are promising candidates for ON delivery to cells because of the following reasons: (1) They are stable for several hours in solutions of up to physiological ionic strength; (2) they are efficiently taken up by cells; (3) after cellular uptake, they easily release the ONs even when these are present as phosphorothioates. D 2005 Elsevier B.V. All rights reserved. Keywords: Nanoparticles; Phosphorothioates; Protamine sulfate; Human serum albumin; Cellular release

1. Introduction The packing of DNA or oligonucleotides (ONs) into nanoparticles to improve the stability, targeting, and transfection efficiency of the molecules is of paramount importance (for reviews, see Refs. [1–3]). * Corresponding author. E-mail address: [email protected] (J. Kreuter). 0168-3659/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2005.04.019

This objective can be reached using nanoparticulate systems, including nanoparticles prepared by selfassembly of ONs and protamine [4–7]. These spherical nanoparticles, called bproticlesb, led to the increased uptake of the ONs by several types of cells, as compared to naked ONs [4–6]. Release of the ONs from the proticles after uptake, however, was observed only when the nucleic acid component consisted of oligodesoxynucleotides (ODNs), where-

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as the more stable and hence more useful phosphorothioates (PTOs) were not released from the particles [6]. Very recently, we have described preparation and characterization of improved proticles consisting of PTO, protamine (free base), and human serum albumin (HSA) as a third component in the starting mixture [8–10]. These bternaryQ proticles share two important advantages with the previously studied bbinaryQ ones: the ease with which they can be prepared and the low costs of the excipients. They are superior to the binary ones in two important aspects: (1) They are stable in solutions of up to physiological ionic strength for several hours, and (2) after uptake by murine fibroblasts, they show ON release also when the compound is present as PTO [8]. The ternary proticles thus seem to deserve serious attention as a potential drug delivery system. As studied in detail recently, the ternary proticles have a broad size distribution, the most abundant diameter being around 200 nm. During sample preparation for transmission electron microscopy (TEM) they were easily dissociated into much smaller spheres, frequently as small as around 30 nm. We therefore concluded that the 30 nm-spheres may represent belementaryQ proticles constituting the building blocks of the larger ones [8,9]. A similar conclusion was drawn from the finding by atomic force microscopy (AFM) that particles of this size occurred during the growth phase of the binary proticles [7]. When we extended our recent experiments on the PTO/protamine free base/HSA proticles by substituting protamine free base by protamine sulfate, we found that the latter proticles predominantly consisted of particles of a diameter around 40 nm. These particles are thus only slightly larger than the previously hypothesised belementaryQ proticles [8]. Our study, which includes experiments on particle uptake and intracellular release by murine fibroblasts, is described below.

2. Materials and methods 2.1. Materials The PTOs consisting of 20 nucleotides as well as the fluorescein isothiocyanate-(FITC)-labelled analoga were purchased from MWG Biotech (Ebersberg,

Germany). For details including their sequence see [7]. Protamine sulfate derived from salmon, HSA (fraction V), and tetramethylrhodamine isothiocyanate-(TRITC-) conjugated concanacalin A were obtained from Sigma-Aldrich (Taufkirchen, Germany). Fetal calf serum (FCS) was from Biochrom (Berlin, Germany), and minimum essential medium (MEM) Eagle from PAA (Co¨lbe, Germany). FITC and TRITC were purchased from Molecular Probes/ MoBiTec (Go¨ttingen, Germany). Water was taken from a Millipore Milli-Q Plus system (Schwalbach, Germany). All other reagents were obtained from Sigma-Aldrich. 2.2. Nanoparticle preparation Ternary PTO/protamine sulfate/HSA proticles were prepared as described for the corresponding proticles made with protamine free base [8]. The protamine sulfate/HSA solution (in buffer) used per experiment had a volume of 0.8 ml and contained 60 Ag protamine sulfate (if not stated otherwise) and 500 Ag HSA. This solution was incubated for 5–10 min and then mixed by vigorous stirring for 5 s using 30 Ag PTO in 0.2 ml water. In most experiments the final buffer composition was 10 mM HEPES (pH 7.0), 150 mM NaCl, 0.1 mM EDTA. For the cell culture experiments, 100 Al of 50 mM HEPES (pH = 7.4), 15 mM NaCl containing 30 Ag FITC-labelled oligonucleotides were added to 900 ml of the same buffer containing 75 Ag protamine sulfate and 100 Ag HSA, and the mixture was stirred for 5 s. It was added to the cells 2 h after mixing the components. 2.3. Sedimentation velocity analysis The sedimentation velocity experiments used a Beckman Optima XL-A analytical ultracentrifuge as described previously [7,8], with rotor speeds of 4000 or 5000 rpm and data collection at 310 nm. The evaluation applied the ls g*(s) variant of the programs sedfit and sedphat of P. Schuck [11–13]. Transformations from distributions g*(s) to the corresponding diameter distributions d*(s) were based on a partial specific volume, v¯, of the ternary proticles of (0.66 F 0.05) ml/g [8].

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2.4. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) For TEM a Philips EM 208S microscope was used and operated at 100 kV. Samples were negatively stained with 1% ammonium molybdate as described recently [14]. AFM imaging of the proticles was performed in liquid environment and under minimal force conditions in tapping mode on a Multimode SPM (Veeco Instruments, Santa Barbara, CA, USA). Triangular silicon nitride cantilevers from Olympus (0.08 N/m; OMCL-TR400PSA, tip radius ~20 nm) were used. Samples were prepared by immersing cantilever and mica substrate in the fresh native undiluted proticle solution. Lateral tip convolution effects cannot be excluded because the tip radius is in the same order of magnitude as the proticles. Therefore, the diameter of the proticles was estimated from their observed height which is not subject to tip convolution effects. 2.5. Cell culture experiments and confocal laser scanning microscopy L(tk-) mouse fibroblasts (American Type Culture Collection CCL 1.3) were grown and used as in [8]. FITC-labelled oligonucleotides were employed in the uptake and release experiments. Cell membranes were stained with TRITC-conjugated concanavalin A. The fixed and embedded cells were viewed in a confocal microscope, and confocal sections were recorded and rearranged to a section view or composed into a threedimensional picture [8].

3. Results bTernaryQ proticles assembled from PTO, protamine free base, and HSA are relatively stable even when formed in solutions of up to physiological ionic strength, in contrast to the corresponding bbinaryQ proticles lacking HSA [8]. Analysis of the distribution brelative abundance versus sedimentation coefficient s 20,wQ, g*(s), measured 2–6 h after mixing the components showed broad curves centered around s 20,wvalues of 10,000 – 15,000 S. Transformation of the g*(s) curves into the corresponding brelative abundance versus diameterQ distributions, g*(d), based on

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the spherical shape of the particles found by EM and assuming that the spheres are solid, yields curves with maxima at d-values around 200 nm [8]. As we replaced protamine free base by protamine sulfate in preparing the ternary proticles, we found the size distributions of the particles to be surprisingly different from those with protamine free base (see below). 3.1. Sedimentation behaviour and corresponding size distributions Ternary proticles containing protamine sulfate are rather stable even when assembled at physiological ionic strength in contrast to those containing protamine free base, which are most stable after formation at about half of that ionic strength [8]. This is demonstrated by the g*(s) distributions of Fig. 1A which show that the peak region of the distributions is virtually unchanged between 2 h and 8 h after mixing the components and only slightly shifted towards higher s-values after 24 h (there is, however, some increase in the abundance of more rapidly sedimenting particles after this time although this was less then with those described previously [8]; this indicates that aggregation occurred in parts of the proticle population). The increased stability as compared to the proticles prepared using protamine free base is demonstrated by Fig. 1B which shows the dependency on time of the relative s-value of the maxima of the respective g*(s) curves for the different particles. The most drastic change between the present and the previous particles is, however, observed with respect to the absolute value of s 20,w: it is now in the range 800–1200 S and thus lower than before [8] by more than a factor of 10. The g*(s) curves were virtually independent of ionic strength I (I = 30–150 mM). The decrease in the average s-value of the particles following replacement of protamine free base by protamine sulfate is accompanied by a decrease in sample turbidity: under comparable conditions, the turbidity values reach only 40–60% of those of the former particles (data not shown). The influence of the PTO/protamine weight ratio in the starting mixtures was found to be somewhat larger than with proticles containing protamine free base [8]. The narrowest distributions were obtained for a 1 : 2 ratio, whereas at ratios of 1 : 3 and 1 : 4 the g*(s) distributions became bimodal, with an additional max-

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A

dant diameter values of particles assembled by the use of protamine free base are around 200 nm (Fig. 2). 3.2. Morphology and size according to TEM and AFM

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Fig. 1. (A) Sedimentation velocity analysis of PTO/protamine sulfate/HSA proticles: g*(s) distributions obtained 2 h ( ____ ), 8 h ( _ _ _ ), and 24 h ( ...... ) after initiating particle formation. Buffer: 10 mM HEPES (pH 7.0), 150 mM NaCl, 0.1 mM EDTA. Rotor speed: 5000 rpm. (B) Time course of the s 20,w-value of the peak of g*(s), as compared to the value after 2 h, for the present proticles and those from Ref. [8]: PTO/protamine sulfate/HSA proticles under the conditions of A (E); PTO/protamine free base/HSA proticles under the same conditions (o) and in 20 mM sodium phosphate (pH 7.0), 10 mM NaCl, 0.1 mM EDTA (5). The latter two curves are derived from the data in Fig. 4 of Ref. [8].

imum around 2000 S. The position of the main peak of g*(s), however, was virtually unchanged. No significant influence of the PTO/protamine ratio concerning changes with increasing time was observed (data not shown). Assuming that the particles are spherical (see below) and solid and applying a value for their partial specific volume v¯ of 0.66 ml/g [8], the g*(s) curves of Fig. 1A obtained 2 h and 24 h after initiating particle formation were transformed into the corresponding diameter distributions g*(d) (Fig. 2). The peak values are 43 and 48 nm, respectively, the half-widths of the curves F 17 and F 19 nm. In contrast, the most abun-

With PTO/protamine free base/HSA proticles the average particle size according to TEM was distinctly smaller in most cases than expected from sedimentation velocity analysis, despite the fact that the results from the latter technique were based on the solid sphere model and thus should represent only lower limit values [8]. Dissociation of the proticles during preparation for TEM and also for AFM, in particular following drying of the particles or the influence of the staining reagents, was shown to be responsible for this discrepancy [8]. The effect seems to be present also with the PTO/protamine sulfate/HSA proticles but with strongly reduced magnitude: the virtually perfectly spherical particles shown in Fig. 3A have a size distribution which overlaps with that given in Fig. 2 but has a maximum at around 30 nm instead of 43 nm (Fig. 3B). The apparent size difference cannot be explained by shrinking of the proticles during drying and staining in preparation for electron microscopy (since a solid sphere should be unable to shrink), nor by use of an inappropriate v¯-value during transformation of the g*(s) curve since v¯ = 0.49 ml/g, which is an unreasonable value [15], would be required to yield a g*(d) curve conforming with the TEM data. Thus it 1.0

g*(d) [a.u.]

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Fig. 2. Transformation of g*(s) data into diameter distributions g*(d), based on a solid sphere model. The g*(s) curves converted are those from Fig. 1A obtained 2 h ( ____ ) and 24 h ( ...... ) after mixing the components, and from Fig. 4A of Ref. [8], obtained with PTO/protamine free base/HSA mixtures 5 h after mixing ( _._._ ). All transformations were based on v = 0.66 ml/g, and the curves were normalised to the same integral.

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d [nm] Fig. 3. Morphology and size of PTO/protamine sulfate/HSA proticles according to TEM. (A) Electron micrograph of a sample which was negatively stained (for 10 s from underneath the grid) 2 h after mixing the components. Buffer: see Fig. 1. Bar: 200 nm. (B). Histogram showing the size distribution n(d) of 705 particles. The smooth curve represents the corresponding size distribution according to analytical ultracentrifugation from Fig. 2.

has to be concluded that drying and/or staining promotes dissociation of the proticles, similarly to those containing protamine free base. This conclusion is supported by AFM results on proticles imaged in solution (Fig. 4): these images yield an average par-

Fig. 5. Uptake of PTO/protamine sulfate/HSA proticles containing FITC-labelled PTO into murine fibroblasts and intracellular localization of the PTO (green), according to confocal laser scanning microscopy. The cell membranes were stained red. (A) Section view; (B) 3-D picture. Bars: 2 Am.

ticle diameter of 40 nm (n = 92), which is very similar to the results from sedimentation velocity analysis applying the solid sphere model. A detailed compar-

Fig. 4. AFM images of the PTO/protamine sulfate/HSA proticles, obtained 2 h after mixing the components. Buffer: see Fig. 1. The image on the right is a zoom of the part marked by a square in the left image. In both images the bar represents 500 nm.

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ison of the size distributions with those from analytical ultracentrifugation or TEM, however, does not seem to be reasonable, due to the small number of particles measured by AFM. An interesting though yet incomprehensible detail in the AFM images was the absence of particles of diameter around or smaller than 10 nm, which were abundant when protamine free base was used for proticle preparation. 3.3. Uptake of the particles by murine fibroblasts and intracellular release of the PTO The bbinaryQ proticles can be taken up by a variety of different cells [4–6]. Intracellular release of the ON component, however, has only been observed with ODNs but not PTOs [6]. In contrast, the ternary PTO/protamine free base/HSA proticles were shown to release the PTOs after uptake by murine fibroblasts [8]. Proticle uptake and subsequent PTO release was also found in the present study, with the much smaller proticles containing protamine sulfate. This is shown in Fig. 5, PTO release from the particles being demonstrated by the nearly uniform FITC staining of the cell interior. As with the PTO/protamine free base/HSA proticles, the efficiency of proticle uptake by the cells is close to 100%.

4. Discussion Studying the size distribution of nanoparticles is an important but intricate task. The main techniques used are various types of microscopy (in particular TEM and AFM), dynamic light scattering (DLS), and sedimentation velocity analysis in the analytical ultracentrifuge. With stable and relatively uniform particles, the three techniques are expected to yield virtually identical results, as confirmed recently by our group [16]. With other samples, the situation can be much more complex and less favourable. E. g., the preparation procedures required for TEM may lead to disintegration of the particles under study, as in the case of the present samples. DLS is nondisturbing with respect to particle structure but with strongly heterogeneous samples lacks resolution. In addition, in the presence of moderate or even small amounts of large particles DLS may overlook particles of distinctly smaller size even if they represent the main compo-

nent ([17]; see also the findings and discussion in [14]). Since strong size heterogeneity is a striking property of all proticles studied by us up to now, we have, in the present study, done without DLS. Sedimentation velocity analysis, in combination with modern software [11–13], is the most reliable of the three techniques, and in the present study we have applied it intensely. As shown by us recently, however, it may overlook small amounts of particles which are much larger than the bulk of the material [8,14]. Our findings on proticles containing protamine free base [8] suggest that this problem could also have affected the present results. With respect to the nanoparticles studied by us there is evidence that, in devising drug delivery particles, substitution of protamine free base by protamine sulfate can be advantageous due to an improved particle uptake by cells [18,19]. After completion of our study on the ternary proticles containing protamine free base [8] analogous experiments were therefore performed in which the latter compound was replaced by the former one. To our surprise, the protamine sulfate particles yielded a different size class, with most abundant diameters around 40 nm instead of 200 nm for the particles containing protamine free base, despite the similarity in their composition. The average diameter of the protamine sulfate particles amounts to only around 20% and thus their average volume to about 1% that of the particles containing protamine free base. Another difference was observed with respect to particle stability with increasing time, which was higher with the protamine sulfate particles (Fig. 1B). No differences were found concerning particle uptake by murine fibroblasts and subsequent release of the PTO component: both kinds of proticles were efficiently taken up by the cells, and both showed intracellular release of the active agent. This similarity, however, may be absent with other types of cells. In such a case it could be advantageous to have two different size options. As already stated in the introduction, during sample preparation for TEM those proticles containing protamine free base with an average diameter of approx. 200 nm easily disintegrate into particles of diameters down to 25–30 nm [8]. Particles of similar sizes also were found as intermediates during the growth of binary proticles [7]. This led us to the hypothesis that particles with a diameter of around

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30 nm may represent belementaryb proticles which then constitute the building blocks of the larger ones [7,8]. In the present paper, particles seemingly identical to these elementary proticles represent part of the proticle population, and others – the major part – seem to be made up of only a few elementary ones. This strongly supports the suggested mode of assembly. Apparently, the elementary PTO/protamine sulfate/ HSA proticles are less prone to aggregation than their counterparts containing protamine free base. The molecular basis for the difference in bstickinessQ between the two kinds of elementary proticles, however, is still unknown to us.

[8]

[9]

[10]

[11]

Acknowledgements We are grateful to the German Bundesministerium fu¨r Bildung und Forschung (BMBF) (project 03C0308A) and the NWO for financial support.

References [1] S.C. De Smedt, J. Demeester, W.E. Hennink, Cationic polymer based gene delivery systems, Pharm. Res. 17 (2000) 113 – 126. [2] I. Lebedeva, L. Benimetskaya, C.A. Stein, M. Vilenchik, Cellular delivery of antisense oligonucleotides, Eur. J. Pharm. Biopharm. 50 (2000) 101 – 119. [3] F. Shi, D. Hoekstra, Effective intracellular delivery of oligonucleotides in order to make sense of antisense, J. Control. Release 97 (2004) 189 – 209. [4] M. Junghans, J. Kreuter, A. Zimmer, Antisense delivery using protamine–oligonucleotide particles, Nucleic Acids Res. 28 (2000) e45. [5] M. Junghans, J. Kreuter, A. Zimmer, Phosphodiester and phosphorothioate oligonucleotide condensation and preparation of antisense nanoparticles, Biochim. Biophys. Acta 1544 (2001) 177 – 188. [6] N. Dinauer, D. Lochmann, I. Demirhan, A. Bouazzaoui, A. Zimmer, A. Chandra, J. Kreuter, H. von Briesen, Intracellular tracking of protamine/antisense oligonucleotide nanoparticles and their inhibitory effect on HIV-1 transactivation, J. Control. Release 96 (2004) 497 – 507. [7] D. Lochmann, V. Vogel, J. Weyermann, N. Dinauer, H. von Briesen, J. Kreuter, D. Schubert, A. Zimmer, Physicochemical

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

187

characterisation of protamine–phosphorothioate nanoparticles, J. Microencapsul 21 (2004) 625 – 641. V. Vogel, D. Lochmann, J. Weyermann, G. Mayer, C. Tziatzios, J.A. van den Broek, W. Haase, D. Wouters, U.S. Schubert, J. Kreuter, A. Zimmer, D. Schubert, Oligonucleotide-protaminealbumin nanoparticles: preparation, physical properties and intracellular distribution, J. Control. Release 103 (2005) 99 – 111. D. Lochmann, J. Weyermann, Ch. Georgens, R. Prassl, A. Zimmer, Albumin-protamine-oligonucleotide nanoparticles as a new antisense delivery system: Part 1. Physicochemical characterisation, Eur. J. Pharm. Biopharm. 59 (2005) 419 – 429. J. Weyermann, D. Lochmann, Ch. Georgens, A. Zimmer, Albumin-protamine-oligonucleotide nanoparticles as a new antisense delivery system: part 2, cellular uptake and effect. Eur. J. Pharm. Biopharm. (in press). P. Schuck, P. Rossmanith, Determination of the sedimentation coefficient distribution by least-squares boundary modelling, Biopolymers 54 (2000) 328 – 341. P. Schuck, Size distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modelling, Biophys. J. 78 (2000) 1606 – 1619. P. Schuck, M.A. Perugini, N.R. Gonzales, G.J. Howlett, D. Schubert, Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems, Biophys. J. 82 (2002) 1096 – 1111. V. Vogel, J.-F. Gohy, B.G.G. Lohmeijer, J.A. van den Broek, W. Haase, U.S. Schubert, D. Schubert, Metallo-supramolecular micelles: studies by analytical ultracentrifugation and electron microscopy, J. Polym. Sci., A, Polym. Chem. 41 (2003) 3159 – 3168. H. Durchschlag, Specific volumes of biological macromolecules and some other molecules of biological interest, in: H.J. Hinz (Ed.), Thermodynamic Data for Biochemistry and Biotechnology, Springer-Verlag, Berlin, 1986, pp. 45 – 128. A. Bootz, V. Vogel, D. Schubert, J. Kreuter, Comparison of scanning electron microscopy, dynamic light scattering and analytical ultracentrifugation fort he sizing of poly(butyl cyanoacrylate) nanoparticles, Eur. J. Pharm. Biopharm. 57 (2004) 369 – 375. B. Berne, R. Pecora, Dynamic Light Scattering — with Applications to Chemistry, Biology, and Physics, Dover Publications, Mineola, NY, 2000. F.L. Sorgi, S. Bhattacharya, L. Huang, Protamine sulfate enhances lipid-mediated gene transfer, Gene Ther. 4 (1997) 961 – 968. S. Li, L. Huang, Protamine sulfate provides enhanced and reproducible intravenous gene transfer by cationic liposome/ DNA complex, J. Liposome Res. 7 (1997) 207 – 219.