Microscopic Investigations into PEG−Cationic Polymer-Induced DNA

Moreover, AFM allows both structures and processes over time to be .... Cross-sectional measurements were taken at the positions displayed in part c a...
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Langmuir 2001, 17, 3185-3193

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Microscopic Investigations into PEG-Cationic Polymer-Induced DNA Condensation Benjamin J. Rackstraw,† Alison L. Martin,‡ Snjezana Stolnik,*,† Clive J. Roberts,‡ Martin C. Garnett,† Martyn C. Davies,‡ and Saul J. B. Tendler‡ Advanced Drug Delivery Group and Laboratory of Biophysics and Surface Analysis, School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, U.K. Received October 17, 2000. In Final Form: February 27, 2001 The morphology of condensates formed between plasmid DNA and a PEG-poly(amidoamine) triblock copolymer were investigated using TEM and AFM in aqueous conditions. Both techniques revealed the presence of ring and extended linear structures; however, a greater proportion of linear structures was obtained with the PEG copolymer than was seen with the poly(amidoamine) homopolymer, indicating that the PEG was interfering with condensation. Differences in the ring diameters between the PEG copolymer and the homopolymer were more marked with TEM than AFM, suggesting that compact toroidal structures are largely due to constriction on dehydration and that this is prevented from occurring in the presence of PEG. AFM revealed an increase in condensate size with increasing polymer/DNA ratio, which can be attributed to the association of more polymer with the complex. With TEM, the proportion of linear structures increased with increasing polymer/DNA ratio, whereas it remained relatively constant for AFM across all ratios. It is suggested that an increased amount of PEG associated with the complexes promotes disruption of the ring structure upon dehydration. This supports the hypothesis that rings are formed via the bending of linear structures, with an equilibrium existing between the two, that is altered in favor of the linear structure by PEG.

Introduction DNA condensation by multivalent cations and polycationic agents has been investigated extensively since the mid-1960s. Early work focused on understanding how DNA is packaged within cell nuclei and viral particles.1,2 Recently, however, interest in the phenomenon has been generated by the advent of the concept of gene therapy and the vast number of diseases that might be treated by this approach.3 The drive to achieve effective gene therapy has resulted in a large-scale search for suitable vectors.4 A significant amount of gene delivery research is now concentrating on polycationic agents as potential vectors. For many of the cationic polymer systems under investigation, the colloidal stability of the complexes formed is not wholly satisfactory,5 and potential problems with biocompatibility and biodistribution arise on transfer from in vitro to in vivo conditions.6 A possible solution has been proposed from experience with nanoparticulate drug delivery systems, for which it has been shown that both the colloidal stability and biodistribution can be improved by the inclusion of hydrophilic polymers [mainly poly(ethylene glycol) (PEG)] in the system.7,8 These polymers are intended to form a steric barrier around the particle, * To whom correspondence should be addressed. Tel.: +44 (0)115-846-6074. Fax: +44 (0)115-951-5102. E-mail: [email protected]. † Advanced Drug Delivery Group. ‡ Laboratory of Biophysics and Surface Analysis. (1) Leng, M.; Felsenfeld, G. Proc. Natl. Acad. Sci. U.S.A. 1969, 56, 1325. (2) Laemmli, U.K. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 4288. (3) Ledley, F. D. Pharm. Res. 1996, 13, 1595. (4) Garnett, M. C. Crit. Rev. Ther. Drug Carrier Syst. 1999, 16, 147. (5) Tang, M. X.; Szoka, F. C. Gene Ther. 1997, 4, 823. (6) Plank, C.; Mechtler, K.; Szoka, F. C.; Wagner, E. Hum. Gene Ther. 1996, 7, 1437. (7) Stolnik, S.; Dunn, S. E.; Garnett, M. C.; Davies, M. C.; Coombes, A. G. A.; Taylor, D. C.; Irving, M. P.; Purkiss, S. C.; Tadros, T. F.; Davis, S. S.; Illum, L. Pharm. Res. 1994, 11, 1800. (8) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263, 1600.

preventing self-aggregation and reducing opsonisation of the particles by the immune system, so acting to improve biocompatibility and increase their circulating life. This principle has been adapted for the purposes of DNA delivery with the development of cationic hydrophilic block copolymers,9 which possess at least one cationic segment that interacts electrostatically with DNA to condense it into a core that is surrounded by a corona produced by the hydrophilic block(s). Our research into cationic polymer agents for the condensation and delivery of DNA has been focused on the linear poly(amidoamine)s, with a range of compounds having been subjected to physicochemical and biological characterization.10,11 Successful transfection of cultured cell lines was demonstrated by a number of these structures, with comparably favorable cytotoxicities.11 From these studies, the most promising candidate was the polymer NG49, which is based on a methylene-bisacrylamide dimethylethylenediamine (MBA DMEDA) repeat unit (Figure 1, structure I). Based on these findings and adopting the PEG steric stabilization approach, the ABA block copolymer PEG-poly(amidoamine)-PEG (NG47) (Figure 1, structure II) was synthesized with the same repeat-unit structure as I and with PEG blocks of 2000 Da. Investigations into the biological performance of this polymer, however, revealed it to be greatly inferior to the parent polymer in terms of both transfection ability and protection of DNA from degradation by nuclease enzymes.12 It was proposed that this deficiency was due to the PEG segments interfering with the interaction of (9) Seymour, L. W.; Kataoka, K.; Kabanov, A. V. In Self-Assembling Complexes for Gene Delivery: From Laboratory to Clinical Trial; Kabanov, A. V., Felgner, P. L., Seymour, L. W., Eds.; John Wiley & Sons: New York, 1998; p219. (10) Jones, N. A.; Hill, I. R. C.; Stolnik, S.; Bignotti, F.; Davis, S. S.; Garnett, M. C. Biochim. Biophys. Acta 2000, 93458, 1. (11) Hill, I. R. C.; Garnett, M. C.; Bignotti, F.; Davis, S. S. Biochim. Biophys. Acta 1999, 1427, 161. (12) Rackstraw, B. J.; Garnett, M. C.; Stolnik, S.; Davis, S. S. Proc. 4th Int. Symp. Polym. Ther. 2000, 68.

10.1021/la001456x CCC: $20.00 © 2001 American Chemical Society Published on Web 05/03/2001

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Rackstraw et al. Table 1. Polymer Properties Mn Mw polydispersity % PAA pKa1 pKa2

Figure 1. Polymer structures. Structure I is the methylenebis-acrylamide dimethylethylenediamine repeat unit of the cationic homopolymer NG49 and the structure upon which the poly(amidoamine) segment of the PEG-cationic polymer-PEG triblock copolymer II (NG47) is based.

the cationic segment with the DNA, thus compromising the polymer’s condensation ability. The present work investigates the formation of complexes between the homopolymer NG49 and its PEGylated derivative NG47 and plasmid DNA, applying the two complementary imaging techniques of transmission electron microscopy (TEM) and atomic force microscopy (AFM). TEM is a technique that is routinely used to assess the morphology of potential DNA delivery systems. The technique requires the use of a stain, in most cases uranyl acetate, that interacts strongly with nucleic acids and to a lesser extent with positively charged groups.13 However, the interaction of the stain with the neutral PEG chains is negligible, essentially rendering PEG invisible. Furthermore, the ability of PEG to provide a sterically stabilizing layer to particles relies on it being hydrated, and the dehydration required in sample preparation for TEM would cause collapse of the PEG chains. Thus, even if the PEG segments were stained, a true representation of particle morphology in near-natural conditions could not be obtained. In this aspect, AFM holds major advantages over TEM in that less manipulation of the sample is required. The adsorption of the sample onto a substrate is still required for AFM imaging, as it is for TEM, but the staining and drying steps can be eliminated when AFM is performed in a liquid. Moreover, AFM allows both structures and processes over time to be investigated under aqueous conditions, which are more relevant to the natural state or environment. Although it is a comparatively new technique for studying DNA,14,15 AFM has quickly progressed to the point where it has been able to provide much useful information on DNA condensation and has recently been used as a means of visualizing the morphology of systems for DNA delivery.16,17 By performing a parallel study of complex formation and morphology using both techniques and subsequently comparing the results, the extent to which sample preparation affects the complexes seen with TEM can be assessed; AFM analysis of NG47-DNA complexes would also provide information that cannot be derived from TEM studies, especially regarding the behavior of PEG. Experimental Section DNA. The 6 kb plasmid pRSVLuc, containing the firefly luciferase gene, was a generous gift from Cobra Thera(13) Hayat, M. A. Principles and Techniques of Electron Microscopy: Biological Applications; Edward Arnold: London, 1981. (14) Hansma, H. G.; Sinsheimer, R. L.; Li, M. Q.; Hansma, P. K. Nucleic Acids Res. 1992, 20, 3585. (15) Hansma, H. G.; Laney, D. E.; Bezanilla, M.; Sinsheimer, R. L.; Hansma, P. K. Biophys. J. 1995, 68, 1672. (16) Dunlap, D. D.; Maggi, A.; Soria, M. R.; Monaco, L. Nucleic Acids Res. 1997, 25, 3095. (17) Golan, R.; Pietrasanta, L. I.; Hsieh, W.; Hansma, H. G. Biochemistry 1999, 38, 14069.

NG47

NG49

19700 28300 1.438 69 8.01 4.54

19800 28200 1.329 n/a 8.01 4.54

peutics (Keele, U.K.). It was supplied as a 1 mg mL-1 solution in water and was used without further treatment for the TEM experiments. For the AFM work, this solution was further diluted in 10% phosphate buffered saline (PBS) to a concentration of 20 µg mL-1. Cationic Polymer. NG47, a poly(ethylene glycol)-poly(amidoamine) ABA copolymer and its parent poly(amidoamine) homopolymer NG49 were synthesized by Dr. F. Bignotti (University of Brescia, Italy), as previously described.18 Table 1 provides a summary of polymer properties. The polymers were supplied as freeze-dried solids, and prior to use, they were dissolved in water to give solutions with concentrations of 10 and 1 mg mL-1. These concentrations were used for TEM sample preparation; for AFM, the polymer was further diluted with 10% PBS to give a solution such that, when added to the DNA solution, an equal volume was required to prepare samples of a specified polymer/DNA ratio in terms of number of polymer repeat units per DNA nucleotide. The ratios investigated were 1:1, 5:1, and 10:1 in the case of NG47-pRSVLuc condensates and 5:1 for NG49pRSVLuc condensates. Solutions were stored at -20 °C. Transmission Electron Microscopy. Preparation of Polymer-DNA Complexes. A sample of solution containing sufficient polymer to give the required polymer repeat unit/DNA nucleotide ratio was added to plasmid DNA (2.5 µg) in 10% PBS such that the final DNA concentration was 10 µg mL-1. The sample was vortexed briefly and incubated for 30 min at room temperature to allow for complex formation. PBS (0.14 M NaCl, 0.01 M phosphate, pH 7.4) was prepared in water from tablets (Unipath Ltd, Basingstoke, U.K.). Samples were prepared in 10% (v/v) PBS. It was found that, when the samples were prepared in PBS, the TEM grids were obscured by precipitation of the uranyl acetate staining solution with the phosphate as also described in the literature.13 This precipitation was reduced by using PBS diluted 10-fold in water. TEM Sample Preparation. A 10-µL drop of complexcontaining suspension was placed onto a copper grid coated with Pioloform resin (TAAB Laboratory Equipment, Reading, U.K.) and left for 1 min, after which excess liquid was removed by blotting with filter paper. This process was repeated, and after sufficient time for air-drying, the grid was inverted onto a drop of a saturated solution of uranyl acetate in 50% alcohol. The sample was then covered and left for 15 min to stain, before being washed with 50% alcohol and two stages of deionized water, with blotting between each wash. The grid was then allowed to dry in air before imaging was performed. Control grids with polymer solution only and buffer solution only were also prepared and examined. TEM Data Acquisition. Grids were analyzed using a JEOL JEM-1010 TEM[ JEOL Ltd., Welwyn Garden City, U.K.), operating at a voltage of 80 kV. Micrographs were taken at magnifications ranging between 20 000 and 200 000× with a Kodak Megaplus digital camera 1.6i, (18) Bignotti, F.; Ranucci, E.; Ferruti, P. Macromol. Rapid Commun. 1994, 15, 659.

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Figure 2. Schematic representation of the calculation of the volume of a ringlike condensate. (a) AFM image (600 nm2) of a ringlike condensate with a z range of 8 nm. This condensate was formed with pRSVLuc and NG47 at the 10:1 ratio. (b) Representative cross section of such a condensate annotated with the readings taken from it. Cross-sectional measurements were taken at the positions displayed in part c and used to calculate condensate volume with the equation shown above.

using the AnalySIS 3.0 software package. This software was also used to make measurements of the condensate size. Atomic Force Microscopy. Preparation of PolymerDNA Complexes. Initially plasmid pRSVLuc was imaged in the absence of polymer. A plasmid sample diluted to 20 µg mL-1 with 10% PBS as described above was further diluted to 2.5 µg mL-1 in a 10 mM MgCl2, 50 mM Tris HCl, pH 7.4 buffer solution. Three 2-µL aliquots of this solution were spotted onto a 1-cm2 disk of freshly cleaved mica (Agar Scientific, Essex, U.K.), which was incubated for 1 min and then rinsed with four 4-mL aliquots of water and dried under a gentle steam of nitrogen. Polymer-DNA condensates were prepared by addition of 20 µL of polymer solution to 20 µL of DNA solution. Of the resulting solution, 20 µL was spotted as a single aliquot onto a 1-cm2 mica disk. For polymer/DNA ratios of 10:1 and 5:1, bare freshly cleaved mica was used as a substrate. In the case of the 1:1 polymer/DNA ratio, the mica was cleaved and pretreated in a 10 mM NiCl2, 100 mM HEPES buffer solution for 1 h, with excess buffer being blotted prior to deposition of condensate solution. Pretreatment of the mica for the 1:1 ratio was necessary in order to promote electrostatic immobilization between the condensates and the mica. All condensate imaging was conducted under 10% (v/v) PBS. All buffer salts used in the AFM experiments were obtained from Sigma-Aldrich, Poole, U.K. All water used in the TEM and AFM experiments was obtained from an ELGA purification system (resistivity 15 MΩ cm), and buffers were filtered through a 0.2-µm-pore-size filter (Sartorius, Go¨ttingen, Germany) prior to use. AFM Data Acquisition. Topographic data were collected using a Nanoscope IIIa Dimension 3000 atomic force microscope (Digital Instruments, Santa Barbara, CA). All

Figure 3. AFM image of uncondensed pRSVLuc plasmid acquired in air. Scale bar equivalent to 500 nm with a z range of 0.5 nm.

imaging was conducted in tapping mode, with 512 × 512 pixel resolution, at a scan speed of approximately 2 Hz and at ambient conditions. For imaging in air, silicon cantilevers, 125 µm in length, with resonant frequencies in the range of 200-300 kHz were employed (Nanosensors, Germany). When imaging in liquid, thin-armed silicon nitride oxide-sharpened triangular cantilevers were selected, operating at resonant frequencies of approximately 8 kHz. To reduce sample distortion through compression due to the tapping, AFM probe data were acquired at a set point chosen to minimize tip-sample interactions.19 All image analysis was carried out using Nanoscope (19) Chen, X, Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Davies, J.; Dawkes, A. C. Probe Microsc. 2001, 2, 21-29.

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Figure 4. TEM and AFM images obtained with NG47 and pRSVLuc in 10% (v/v) PBS. A and B are TEMs for a 1:1 ratio of polymer repeat units to DNA nucleotides; C is the corresponding AFM image. The images for a 5:1 NG47/pRSVLuc ratio is displayed in the same way in D, E (TEM), and F (AFM). G, H (TEM), and I (AFM) are the images obtained for a 10:1 NG47/pRSVLuc ratio. TEM samples were prepared on Pioloform resin-coated Cu grids and stained with uranyl acetate. For AFM, the 1:1 ratio was prepared by immobilization on NiCl2 pretreated mica, whereas the 5:1 and 10:1 ratios were immobilized on bare mica. All of the AFM scale bars represent 500 nm. Images have a z range of 12 nm.

software. Background slope was removed using a first- or second-order polynomial function. Calculation of Ringlike Condensate Volumes. Structural volumes of the ringlike condensates were determined using Nanoscope software. Cross sections of the ringlike condensates were taken at 45° separations, as shown in Figure 2. Toroidal radius measurements of these condensates were made from the cross sections at half-maximum height in order to minimize tip convolution effects and were used to calculate the condensate volume using the equation

Volume ) 2π2r2R

(1)

where values are as shown in Figure 2. Results TEM and AFM Imaging of Polymer-DNA Condensates. To appreciate the complex architecture of the polymer-DNA condensates, an image of uncondensed

pRSVLuc plasmids has been provided for comparison in Figure 3. This image was acquired in air using a method that is well-established in the AFM imaging of DNA.20 The plasmids display a relaxed, open-loop structure with little twisting or fasciculation of the strands, which is characteristic of uncondensed DNA morphology. Representative TEM and AFM images of NG47pRSVLuc condensates of the polymer repeat unit-to-DNA nucleotide ratios investigated are shown in Figure 4. It is apparent that similar structures are obtained irrespective of the ratio of polymer to DNA with both imaging techniques. They can be broadly divided into two groups; first, the ring structures, which do not appear compact enough to be referred to as toroidal in the strictest sense and so will be referred to as “rings”, and second, structures with a linear morphology, which usually have loops at either end reminiscent of plectonemically supercoiled (20) Wagner, P. FEBS Lett. 1998, 430, 112.

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Figure 5. Images of condensates formed between NG49 and pRSVLuc in a 5:1 polymer repeat unit to DNA nucleotide ratio in 10% (v/v) PBS. The TEM image (A) was prepared on a Pioloform resin-coated Cu grid, and stained with uranyl acetate. The AFM image (B) has the condensates immobilized on bare mica. In this image, the scale bar is equivalent to 500 nm, and the image has a z range of 12 nm. Table 2. Outer Mean Diameters for Ringlike PRSVLuc Condensates with NG47 or NG49 Polymers Measured by TEM and AFM n NG47/pRSVLuc 1:1 NG47/pRSVLuc 5:1 NG47/pRSVLuc 10:1 NG49/pRSVLuc 5:1 NG47/pRSVLuc 1:1 NG47/pRSVLuc 5:1 NG47/pRSVLuc 10:1 NG47/pRSVLuc 5:1

mean ( SD (nm)

100 100 67 130

TEM 76.2 ( 20.5 88.7 ( 20.9 86.6 ( 21.1 59.8 ( 8.0

47 27 35 36

AFM 114.3 ( 19.2 137.6 ( 29.7 149.7 ( 27.1 118.3 ( 19.8

max (nm) 145.8 166.2 156.1 83.1 176.3 202.7 202.0 170.2

Table 3. Comparison of Percentage Occurrences of Rings and Linear Structures for PRVSLuc Condensates with NG47 or NG49 Polymers n

min (nm) 44.7 44.9 53.4 42.4 75.9 78.2 105.5 87.9

DNA. Figure 5 shows images obtained by TEM and AFM of condensates of the parent poly(amidoamine) homopolymer, NG49, with pRSVLuc at a polymer repeat unit/ nucleotide ratio of 5:1. The TEM image contains a number of what could be referred to as “classical” toroidal structures, being much more compact than the rings obtained with NG47, as well as larger, darkly stained entities that are believed to be aggregates of more than one toroid. There are also a smaller number of rodlike structures present. When the ringlike NG49-pRSVLuc condensates are observed with AFM, they do not appear as compact as they do with TEM. Similar linear and aggregated structures are also observed. Measurements of the maximum diameters of the ringlike structures taken from the TEM and AFM images are summarized in Table 2. The diameters and resulting distribution indicate that, for NG47-pRSVLuc complexes, the ring size does not change significantly across the polymer/DNA ratios investigated. In contrast, significantly smaller mean diameters are seen for the more compact NG49 condensates. Although the trends of the TEM and AFM measurements are similar, the absolute AFM values are larger. This difference is attributed, first, to the condensates being solvated structures when investigated by AFM and, second, to the fact that poly(ethylene glycol) does not stain with uranyl acetate so that it cannot be visualized by TEM. Table 3 shows the relative proportions of rings to linear structures. The proportion of linear structures was greater for complexes formed with NG47 than for those produced with NG49. Furthermore, the TEM data show variation

% rings

% linear

NG47/pRSVLuc 1:1 NG47/pRSVLuc 5:1 NG47/pRSVLuc 10:1 NG49/pRSVLuc 5:1

TEM 100 100 67 130

48 42 36 81

52 58 64 19

NG47/pRSVLuc 1:1 NG47/pRSVLuc 5:1 NG47/pRSVLuc 10:1 NG49/pRSVLuc 5:1

AFM 521 405 215 131

42 43 45 76

58 57 55 24

in the proportions of each type of structure across the ratios of NG47, with relatively more rings at 1:1, and the proportion of linear structures increasing with increasing polymer/DNA ratio. This trend is not observed with AFM, where the relative proportion of rings to linear structures across the ratios of NG47 remains constant. However, in accordance with the TEM data ringlike structures are the predominant condensate produced with NG49. Assessment of Ringlike Condensate Volume. AFM captures topographical information in three dimensions; hence surface topography can be analyzed in terms of the x, y, and z axes. This concept is best shown by illustration. In Figure 6, a ringlike structure and a linear structure are presented in a three-dimensional orientation. Analysis of such three-dimensional information allows for the assessment of structural volume, an assessment that is difficult from purely lateral dimensions. The calculated volumes of the ringlike condensates of the NG47/pRSVLuc ratios of 1:1, 5:1, and 10:1 and the NG49/pRSVLuc ratio of 5:1 are presented in Figure 7. For all systems, similar magnitudes of the condensate volume are observed. Focusing first on the NG47 complexes, it is observed that, across the ratios, there is an increase in both the median and the spread of the distribution of the calculated volumes, with the 1: 1 ratio averaging smaller volumes with a reduced distribution. The ringlike structures produced when plasmid DNA is condensed with NG49 display a distribution similar to that of NG47pRSVLuc at the 1:1 ratio. In the cases of the NG47/ pRSVLuc ratios of 5:1 and 10:1, there are two distinct peaks of condensate volumes. In both cases, the condensate volumes of the larger ring structures are approximately double that of the smaller condensates.

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Figure 6. AFM images of a ringlike and a linear condensate viewed in three dimensions to depict the relative volumes of these condensates. Both condensates were formed between NG47 and pRSVLuc at a ratio of 10:1. Images are 500 nm2 with a z range of 8 nm.

Discussion Biological evaluation of the PEG-poly(amidoamine)PEG triblock copolymer, NG47, revealed an inferior performance in terms of ability to protect DNA from degradation by DNase I enzyme and cellular transfection, when compared with the parent poly(amidoamine) homopolymer, NG49.12 Spectroscopic analysis of complexes formed with NG49 and incubated at 37 °C with DNase I revealed no increase in the extinction of UV light at 260 nm, which is associated with degradation of DNA; however, with the corresponding complexes produced with NG47, such an enhancement was observed to occur. The presence of PEG might have been expected to reduce transfection through the shielding of the net positive charge of the polymer-rich complexes and the formation of a steric barrier, preventing contact of the complexes with the cells and reducing their subsequent uptake. However, it was believed that the discrepancy in the level of transfection obtained was not exclusively due to this PEG steric effect, and the loss of nuclease protection seen suggested that the PEG was interfering with the condensation of the DNA by the poly(amidoamine) segment. This disruptive effect of hydrophilic polymer segments on the condensation of DNA by block copolymers has been suggested previously as an explanation for the poor biodistribution behavior of complexes.21 Visualization of (21) Oupicky´, D.; Koa´k, C.; Dash, P. R.; Seymour, L. W.; Ulbrich, K. Bioconjugate Chem. 1999, 10, 764.

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the morphology of the complexes produced by NG47 and NG49 with plasmid DNA seems to confirm that this is the case. From a comparison of the polymer-DNA complex images obtained here by both AFM and TEM (Figures 4 and 5) with those of plasmid DNA alone (Figure 3), it is apparent that DNA condensation does occur to some extent with NG47. However, the images also suggest that the PEG segments are interfering with the formation of compact condensates. The compact toroids and rods that are seen with TEM for NG49 (Figure 5a) are typical in terms of the form and size (toroidal diameter of ca. 60 nm) of the DNA condensates previously observed with cationic polymers.5 In contrast, the structures produced by NG47 are in the form of loose rings and extended, linear plectonemic-like structures, the latter being present in higher proportions than the corresponding population of rodlike particles seen with NG49. Comparable extended structures have been reported by other investigators examining complex formation with copolymer systems. AFM and TEM of PEG-block-PLL22,23 and TEM of PEGgraft-PEI24 revealed condensate morphologies similar to that observed here for NG47. Comparison of these results and those obtained by other researchers highlights the significance of polymer architecture to the morphologies observed. Whereas, with cationic homopolymers, similar condensate structures were observed independently of varying polymer architectures (e.g., linear vs branched),5 the morphology of condensates produced with PEG-blockPLL were seen to differ when linear PLL was used22 compared to when a PLL dendrimer was employed.25 Furthermore, extended condensate morphologies were only observed with PEG-graft-PEI when 2 kDa PEI was used; copolymers produced with 25 kDa PEI yielded fully condensed structures.24 The loose rings and extended plectonemic-like structures observed here are believed to be a consequence of the triblock ABA architecture of NG47 and the central cationic polymer segment being of insufficient size to properly condense DNA, without interference from the attached PEG. Hence, the precise morphologies of the complexes obtained can be supposed to be highly dependent on the structure of the copolymer, specifically the size of the hydrophilic and cationic polymer blocks and their relative proportions, as well as their respective architectures. Previous investigations of the morphology of complexes formed between DNA and copolymers have relied on one or another of the microscopy techniques. A direct comparison of the two techniques provides information on the extent to which sample preparation affects the observed morphology. The structures revealed by each imaging technique are morphologically similar and thus result from the polymer rather than the sample preparation process. However, dehydration of the samples for TEM imaging can be seen to reduce the size of the structures obtained and also to alter the relative proportions of the condensate types. Examination of the condensate diameter measurements taken from images obtained by each technique reveals a more marked difference between NG49 and (22) Wolfert, M. A.; Schacht, E. H.; Toncheva, V.; Ulbrich, K.; Nazarova, O.; Seymour, L. W. Hum. Gene Ther. 1996, 7, 2123. (23) Kwoh, D. Y.; Coffin, C. C.; Lollo, C. P.; Jovenal, J.; Banaszczyk, M. G.; Mullen, P.; Phillips, A.; Amini, A.; Fabrycki, J.; Bartholomew, R. M.; Brostoff, S. W.; Carlo, D. J. Biochim. Biophys. Acta 1999, 1444, 171. (24) Nguyen, H. K.; Lemieux, P.; Vinogradov, S. V.; Gebhart, C. L.; Guerin, N.; Paradis, G.; Bronich, T. K.; Alakhov, V. Y.; Kabanov, A. V. Gene Ther. 2000, 7, 126. (25) Choi, J. S.; Lee, E. J.; Choi, Y. H.; Jeong, Y. J.; Park, J. S. Bioconjugate Chem. 1999, 10, 62.

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Figure 7. Volume of polymer-DNA condensates. Histograms showing the volume distributions of polymer repeat unit-to-DNA nucleotide ratios of (a) NG47/DNA 1:1, (b) NG47/DNA 5:1, (c) NG47/DNA 10:1, and (d) NG49/DNA 5:1 of ringlike condensates imaged with AFM under 10% (v/v) PBS.

NG47 condensates with TEM; the NG49 condensates observed by AFM under aqueous conditions are not dramatically different from the ring structures produced with NG47. It is likely, therefore, that the compact toroids seen with NG49 using TEM are a result of constriction during dehydration. It seems that, with NG47, the presence of PEG does not permit this constriction to occur to the same extent and, thus, the structures remain as loose rings. An explanation for this could be that, at corresponding polymer/DNA ratios, more polymer material is present in NG47 systems because of the presence of PEG. There is, therefore, a greater amount of material associated with the DNA, which, upon dehydration, prevents the full constriction from taking place. From this, it can be hypothesised that the loose rings observed for NG47 by TEM are closer to the actual form of polymerDNA complexes in an aqueous environment than to the more compact toroids seen with superior condensing systems. In other words, the hydrated “natural” form of polymer-DNA condensates is much looser than previously revealed by TEM investigations.5,23,26 The polymer/DNA ratios studied include those where an excess of polymer was present (5:1 and 10:1). Such ratios have shown the best in vitro transfection results, when the systems are formulated with the cationic homopolymer. This is largely due to the increased amount of cationic polymer associated with the complexes, giving them a net positive charge and, hence, increasing their attraction to cells. In the preparation of samples for AFM analysis, one might also predict this to promote condensate (26) Baeza, I.; Gariglio, P.; Rangel, L. M.; Chavez, P.; Cervantes, L.; Arguello, C.; Wang, C.; Montanez, C. Biochemistry 1987, 26, 6387.

adhesion to the negatively charged mica surface. This was indeed observed to be the case for NG47-DNA condensates at polymer-rich ratios where the complexes readily adhered to the mica substrate. In contrast, when bare mica was used as the substrate at the lower NG47-topRSVLuc ratio of 1:1, no complexes were observed. Because the parallel TEM investigation demonstrated the presence of condensates at this ratio, it seems that they were present in the bulk but did not adhere to the untreated mica, suggesting that the complexes do not possess a net positive charge. It is believed that the NiCl2 pretreatment of the mica does not induce condensate formation or affect the observed conformation because, with the experimental protocol adopted, DNA condensation is believed to occur predominantly before the polymer-DNA solution is brought into contact with the mica surface. Additionally, free NiCl2 is not thought to dissociate into the bulk in quantities sufficient to produce what is a gross structural change from loose plasmid DNA to condensate structures. It might be possible that, at higher ratios of polymer to DNA, the excess polymer coats the mica surface, producing a substrate with a net positive charge. Gel electrophoresis (data not shown) has demonstrated that free polymer exists at high polymer/DNA ratios; hence, some coating of the surface might occur. However this coating does not appear to be pronounced, as the mica surface is smooth and free of the small deposits, 2-3 nm in diameter, seen when polymer solution is imaged in the absence of DNA (data not shown). The results obtained here by AFM highlight the increase in the diameter of the rings with increasing polymer/DNA ratio, an increase that is not apparent from the TEM

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Figure 8. TEM images of condensates formed at a NG47/pRSVLuc ratio of 5:1 that seem to suggest ring formation can occur by the bending of linear plectonemic-like structures.

images. This correlates well with the above observations in suggesting that the increase in diameter is due to a greater amount of polymer associated with the complex, but it also points toward the possibility that the increase is a consequence of imaging the hydrated system, where larger amounts of PEG produce more noticeable size increases. Because PEG does not stain and because it collapses on dehydration during TEM sample preparation, these differences are somewhat less appreciable using TEM. The intrinsic ability of AFM to gather topographical information in three dimensions has been exploited to assess the structural volume of polymer-DNA condensates. The median condensate volumes have been shown to be of similar magnitudes for all of the condensate systems studied. The condensate structures produced between NG47 and plasmid DNA have shown an increase in the median value and distribution as the ratio of polymer repeat unit to DNA nucleotide increases. This increase can once again be attributed to the increased quantity of PEGylated polymer associated with the condensates. Complexes formed between NG49 homopolymer and plasmid DNA at a 5:1 ratio are of a magnitude and distribution most similar to those of the NG47/DNA ratio of 1:1. The reduced median and distribution of the NG49 system to the corresponding 5:1 ratio of the NG47 system is due to the physical absence of PEG from the system. On close inspection of Figure 7, it is observed that, especially in the case of the NG47 ratios of 5:1 and 10:1, condensate volumes form two distinct peaks, with the smaller of these two volumes being approximately half that of the greater. This effect is believed to be due to the presence of condensates consisting of one and two plasmids, respectively. The ability of systems with these ratios to form condensates consisting of two plasmids is believed to be due to stretches of cationic polymer not involved in electrostatic interactions with DNA that can bind to another DNA molecule. The physical presence of the PEG, however, might sterically hinder the complexation of a greater number of plasmid molecules. At the 1:1 ratio, there is a reduced amount of this partially uncomplexed polymer present, and so, the larger condensates are not observed. In the case of the NG49 system, multimolecular aggregates are seen, although they are not ringlike. Their tendency to form these aggregates is believed to be due to the absence of PEG. It has already been stated that the linear structures seen with NG47 are present in greater proportions than the rodlike particles with NG49. This is true for all of the polymer/nucleotide ratios studied and for both imaging techniques, suggesting that PEG increases the tendency

toward producing linear structures. No trend is visible across the ratios for AFM, as the linear population remains fairly steady, perhaps indicating that, under aqueous conditions, the system is at equilibrium. However, with TEM, the proportion of linear structures increases with increasing polymer/nucleotide ratio. A possible explanation is that, on dehydration of the samples prior to imaging, the increased amount of PEG associated with rings at higher ratios causes disruption of the rings to yield more of the linear type. This hypothesis is consistent with there being an equilibrium between rings and linear structures under normal conditions and the disruptive effect of PEG on condensation. Taking the above observations into account allows for the development of a theory of condensation that applies to this particular system. First, PEG-mediated disruption of condensation would lead to an increased number of intermediate structures present at any one time, which can be confirmed with TEM, where the prepared samples are “snapshots” of the system. Figure 8 shows a number of condensates that seem to be rings caught at the point of formation via the bending of a linear structure; this phenomenon was captured by TEM several times and implies that this is how the rings are formed. The increased proportion of linear structures suggests that they are actually intermediates in the ring-formation process. This is comparable to the existing theory that toroids are formed from rods,16,27 especially when the increased hydration of structures in natural conditions is taken into consideration, as it is easier to envisage the bending of extended linear structures into rings than the bending of compact, shrunken rods into toroids. The disruption of rings on dehydration, to give linear structures, suggests that this is a reversible process. Further investigations of NG47plasmid DNA systems under aqueous conditions by AFM have been able to detect such an interchange between linear and ring structures taking place.28 This theory can potentially be applied to all cationic polymer systems, with an equilibrium existing between rods or linear structures and toroids or rings. In the absence of PEG, the formation of toroids is favored, so analysis of the populations would reveal a greater proportion of toroids than rods. When PEG is involved, the disrupting effect on condensation shifts the balance toward the linear structures. Under normal aqueous conditions, increasing the amount of PEG polymer in the system does not lead to further ring disruption, as the additional (27) Eickbush, T. H.; Moudrianakis, E. N. Cell 1978, 13, 295. (28) Martin, A. L.; Davies, M. C.; Rackstraw, B. J.; Roberts, C. J.; Stolnik, S.; Tendler, S. J. B.; Williams, P. M. FEBS Lett. 2000, 480, 106.

PEG-Cationic Polymer-Induced DNA Condensation

Langmuir, Vol. 17, No. 11, 2001 3193

polymer is incorporated with the complexes in such a way that the hydrated PEG chains do not interfere with the cationic polymer-DNA condensation any more than they would at lower polymer/nucleotide ratios. However, upon dehydration of the sample for TEM imaging, with more PEG polymer present, the collapse of the PEG provokes further disruption of rings, forming more linear structures.

sible. In terms of DNA delivery, the prospects for NG47 remain poor, but the results observed and the theories derived from them in this work could not have been achieved with a better condensing system. There is a very strong likelihood that the condensation process for this system is the same as that for systems with greater potential for development as DNA delivery vehicles.

Conclusions

Acknowledgment. The authors gratefully acknowledge the EPSRC and Royal Pharmaceutical Society of Great Britain for providing funding for B.J.R. and A.L.M., respectively. We also thank Dr. Fabio Bignotti of the University of Brescia, Italy, for providing the polymers and Dr. Trevor Gray of the Department of Histopathology, Queen’s Medical Centre, Nottingham, for the use of the TEM facilities.

Although, from the results presented here, it is impossible to confirm the sequence of the DNA condensation process that yields the observed structures, the combination of the two imaging techniques and subsequent comparison of their results has provided useful insights into the process of DNA condensation. As the technique of AFM imaging of polymer-DNA systems under aqueous conditions is developed, further elucidation will be pos-

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