Structural Analysis of Chitosan Mediated DNA Condensation by AFM

Biomacromolecules 2004, 5, 928-936

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Structural Analysis of Chitosan Mediated DNA Condensation by AFM: Influence of Chitosan Molecular Parameters Signe Danielsen,† Kjell M. Va˚rum,‡ and Bjørn T. Stokke*,† Biophysics and Medical Technology, Department of Physics, NOBIPOL, Department of Biotechnology, The Norwegian University of Science and Technology, NTNU, NO-7491 Trondheim, Norway Received December 2, 2003; Revised Manuscript Received February 17, 2004

Chitosan is a nontoxic and biodegradable polysaccharide that has recently emerged as a promising candidate for gene delivery. Here the ability of various chitosans, differing in the fractional content of acetylated units (FA) and the degree of polymerization (DP), to compact DNA was studied. Polyplexes made from mixing plasmid DNA with chitosan yielded a blend of toroids and rods, as observed by AFM. The ratios between the fractions of toroids and rods were observed to decrease with increasing FA of the chitosan, indicating that the charge density of chitosan, proportional to (1 - FA), is important in determining the shape of the compacted DNA. The amount of chitosan required to fully compact DNA into well-defined toroidal and rodlike structures were found to be strongly dependent on the chitosan molecular weight, and thus its total charge. A higher charge ratio (+/-) was needed for the shorter chitosans, showing that an increased concentration of the low DP chitosan could compensate for the reduced interaction strength of the individual ligands with DNA. Employing chitosans with different molecular parameters offers the possibility of designing DNA-chitosan polyplexes with various geometries, reflecting various chitosan-DNA interaction strengths, which is necessary for the evaluation of efficient gene delivery vehicles. Introduction The intriguing condensation of DNA has attracted considerable interest for a long time. This is motivated by the search for an understanding of the chromosomal packing of genes, the polyelectrolyte complexation and phase transitions in general, and, more recently, the condensation of gene vectors required for gene therapy. Polyelectrolyte complexes of DNA by different condensing agents typically yield blends of toroids, with diameters in the size range 50-100 nm, and rods.1-3 Alongside the experimental studies, theories for a molecular understanding of the condensation behavior have been proposed. Packing of DNA into a condensed structure involves overcoming the Coulombic barrier related to the negatively charged phosphates on the DNA. Other energetic barriers arise from the loss in configurational entropy when organizing the extended DNA molecule into well-defined structures and the bending of the stiff double helix. Binding of a multivalent cationic ligand to DNA is an exchange reaction where counterions are released both from the DNA and the ligand, causing an increase in the overall entropy.4-6 To overcome the electrostatic repulsion within the DNA molecule, its negative charges must be sufficiently neutralized. Using Manning’s counterion condensation theory, Wilson and Bloomfield showed that 90% of the DNA charge must be neutralized for condensation to occur.7 The details of the condensation behavior of DNA are reported to depend on the polycation.8-11 * To whom correspondence should be addressed. † Biophysics and Medical Technology, Department of Physics. ‡ NOBIPOL, Department of Biotechnology.

Understanding the condensation process is of interest within biotechnology and medicine as it is an important prerequisite for the transport of therapeutic genes toward target cells for gene therapy applications. The compacted form of DNA protects it from nucleases12-14 and, due to reduced dimensions of the molecule, facilitates transport through the extracellular matrix and thus enhances the cellular uptake by the cell through endocytosis. Development of nonviral gene delivery systems has attracted an increasing interest toward tailor-made polycations. These include studies of homopolymeric polycations such as poly-L-lysine (PLL)12,13,15-17 and poly(ethylenimine) (PEI),16,18-21 through other cationic agents such as spermidine,2,7,8,22 cobolthexamine8,23,24 and cationic lipids,19,25 to polycations produced by combinatorial chemistry.11,21,26 PEI is among the candidates that have reported a large application potential due to its high transfection efficiency.16,18 The linear copolymer chitosan has recently emerged as an interesting gene delivery vehicle because of its nontoxic and biodegradable nature.27-29 Further, DNA-chitosan complexes have recently been reported to have high transfection efficiency, comparable to that of PEI.20,29 Chitosan is a partly or completely de-N-acetylated derivative of the naturally occurring polysaccharide chitin, consisting of different compositions of (1f4)-β-linked N-acetylD-glucosamine and D-glucosamine residues. The intrinsic pKa values of the amino groups in the deacetylated units have been determined to be ∼6.5, independent of FA,30,31 and thus give rise to the polycationic behavior of chitosan. The effective charge density of chitosan can be controlled through the preparation of samples with different fractions of the

10.1021/bm034502r CCC: $27.50 © 2004 American Chemical Society Published on Web 04/06/2004

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Chitosan Mediated DNA Condensation

N-acetylated units, FA, as well as by controlling the pH of the solutions. As observed previously,32,33 and also found here, chitosan is an effective agent for DNA condensation. The primary amine of chitosan can also readily be used for grafting ligands for specific cell targeting. Previous studies of condensation behavior of DNA induced by chitosan have included only a limited set of molecular parameters. Here, this is expanded with respect to the degree of acetylation (FA) and the degree of polymerization (DP), and their ability to complex DNA is studied. The charge density of the chitosan molecule is potentially affecting the strength of the interaction with DNA. Since the intersegment interaction strength is shown to be an important factor in influencing the shape of compacted semiflexible polymers,34,35 the charge density of chitosan might affect the structure, dimensions, and stability of the DNA-chitosan polyplexes. The interaction strength within the polyplexes is also identified to be an important factor in controlling the intracellular release of the DNA vector for expression.20,29,32,36 We therefore focused on characterizing the shape and dimensions of the structures formed. The DNA-chitosan complexes were characterized by tapping mode atomic force microscopy (TM-AFM) and then analyzed to yield more detailed information about heights, contour lengths, and geometric structures.

Table 1. Properties of the Various Chitosans Used for DNA Condensation Studiesa

FA 0.01 0.15 0.35 0.49

[η] (ml/g) 220 610 42 740 68 760 100 450

K 0.343 0.100 1.72 × 10-2 5.02 × 10-3

a 0.624

term used Mn ∼DP (kDa) for identification

198 1004 0.731 24 1171 0.883 68 1036 0.989 121 562

32 162 4 196 12 182 22 102

C(0.01,32) C(0.01,162) C(0.15,4) C(0.15,196) C(0.35,12) C(0.35,182) C(0.49,22) C(0.49,102)

a The number average molecular weight, M , was calculated using the n MHKS equation. K and a refer to the constants in the MHKS equation.

concentration, cchit. The DNA stock solution was diluted in NH4Ac (150 mM, pH 7.4), and complexes were then prepared by adding the chitosan solution to the DNA solution, yielding a final DNA concentration, cDNA, of 3-6 µg/mL and average charge ratios ([qchit]/[qDNA]) in the formulation between 1 and 6 in different experimental series. The relation between the DNA to chitosan concentration ratio (cDNA/cchit) and the charge ratio k () [qchit]/[qDNA]), taking into account the actual pH in the solution, is given by (counterions not explicitly included):

Materials and Methods

cDNA 1 Mw(DNA) (1 - FA) ) cchit k qDNA (42F + 161)(1 + 10pH - pKa) A

Samples. The plasmid DNA pBR322 (4363 bp, Bo¨rhing Mannheim) was employed in most experiments. From standard absorption spectroscopy methods, measuring the OD260/OD280 ratio, the protein content in the solution was determined to be zero. The pBR322 was therefore used as provided. Additional experiments were performed with a linear DNA (D1626, ∼2000 bp, Sigma) to investigate possible influence of linear or circular DNA on the type of complexes being formed. A stock solution (1 mg/mL) of the linear DNA was prepared in MQ water. Eight different chitosans, varying in the degree of acetylation (FA) and the degree of polymerization (DP), were used. The chitosans were prepared by heterogeneous de-N-acetylation of chitin. The acid-soluble fraction was prepared as previously described37 and subsequently converted to the corresponding hydrochloric salts.38 Low molecular weight chitosans were prepared by nitrous acid depolymerization and subsequent reduction with NaBH4.38 The molecular mass of the various chitosans were estimated from the experimentally determined intrinsic viscosity, [η], using the Mark-Houwink-Kuhn-Sakurada equation (MHKS), where the constants K and a were estimated for each FA according to Anthonsen et al.39 Table 1 lists the properties of the various chitosans. We adapt the sample notation C(FA, Mn) from Schipper et al.40 where the first number in the bracket reflects the FA and the second number reflects the number average molecular weight in kDa. Complex Formation and Sample Preparation for AFM. Chitosan stock solutions (0.2-1 mg/mL) were prepared in 1% acetic acid and further diluted in ammonium acetate (NH4Ac, 150 mM, and pH 7.4) to the selected chitosan

where Mw(DNA)/qDNA ) 330 g/mol‚e is the mass per unit charge of DNA, and the pKa of chitosan is equal to 6.5.30,31 All experiments were performed at pH 7.4 and at an ionic strength (I) in the solution of 150 mM. Ammonium acetate was used, instead of the more commonly used nonvolatile salts, e.g., NaCl, to adjust the ionic strength in the solutions to avoid formation of salt crystals in the dried specimens and associated obscuring effects in the images.41 The cDNA/ cchit ratios used to obtain k ) 1 at pH 7.4 corresponds to a ratio between the number of amine groups and phosphate groups equal to 9 (or k ) 4.5 at pH 6.5), which is in the range used for transfection studies.20 Samples for AFM imaging were prepared by depositing an aliquot of 10 µL of the aqueous specimen on a freshly cleaved 5 mm diameter mica surface. After an ∼2 min incubation on the mica disk, the samples were dried with a stream of N2 gas and then further vacuum-dried (P ∼ 10-4 Pa) for at least 2 h. Uncomplexed DNA was immobilized on the mica surface using 0.5 mM ZnCl242 added to the solution immediately before transferring the solution to the mica and then dried as described for the DNA-chitosan complexes. AFM Imaging and Image Analysis. The dried specimens were imaged by tapping mode AFM (TM-AFM) in air using a Digital Instrument Multimode IIIa equipped with an E scanner (maximum xy range ∼15 µm) as described previously.43 The topographs were obtained at scan sizes in the range 1.5-2.5 µm (data collection at 512 × 512 pixels) and with a scan speed of approximately 1.5 Hz. Quantitative determination of the geometries of the DNAchitosan complexes and their distributions were carried out

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by image analysis of the AFM height topographs as detailed previously43 using a software developed in the IDL language (Research System, Inc.). The structures were sorted into three different classes, linear, toroidal, and globular, based on the calculation of a shape factor, the asphericity index A, for each structure.35,43 This asphericity index reflects the symmetry of the structure about the three axes of rotation. Theoretical values of the asphericity index are A ) 0 for a sphere, A ) 0.25 for a circle without any width, and A ) 1 for a rod without any width.35 Complexes were included in the toroidal ensemble provided A ∈ (0.2-0.35) and using the additional criteria of a distinct hole in the center. The ensemble of topologically linear polyplexes included species with A ∈ (0.5-1.0) to also allow for curved species. The structures were analyzed to extract contour length and height data within each ensemble. A routine adapted from Hoh and co-workers,44 involving thinning of the structures with backextrapolation of the pixels at the ends, was used to analyze the linear structures. The height along the contour length of the thinned images was used as the height of the morphological linear structures. For the toroids, cross-sections were drawn radially from the center of mass at incremental angles of 4°, giving the height profile of the species. The contour lengths of the toroids were obtained from the maximum heights in the cross-sections collected for the individual toroids. This method allows the height of the structures to be measured continuously along the contour length and thus height differences within each complex to be determined. Globular structures were characterized with the volume contained within a perimeter of the half-height of the maximum. The number of species included in each ensemble for the quantitative determination of the dimensions was in the range of 50-100. Results and Discussion Observation of DNA-Chitosan Complexes, Plasmid Versus Linear DNA. Complexation of both linear and circular DNA with chitosan yields a blend of toroidal and rodlike structures (Figure 1). These morphologies are also typical when DNA is complexed with other condensing agents, i.e., PLL,12,15,45 PEI,46,47 and cobolthexamine.8,24,48,49 Even though the formation of the rodlike and toroidal structures was seen to be independent upon whether a linear or a circular DNA is used, the ratio between the observed number of toroids and rods was found to differ. Complexation with the chitosan C(0.01,162) at k ) 1 yielded reduction in the toroid-to-rod ratio from 2.3 for the plasmid to 1.6 for complexation using the linear DNA. Furthermore, a larger fraction of globular structures was observed when using the linear DNA rather than the plasmid DNA. Analyzing the dimensions of complexes also revealed that both the height and contour length distributions are broader when using the linear DNA instead of the plasmid (data not shown). The differences in the structures formed from the two DNAs could at least partly arise from the polydispersity of the employed linear DNA. It has been reported that the chain length of DNA influences its ability to form well-defined toroidal and rodlike structures as well as the relative

Danielsen et al.

Figure 1. Tapping mode AFM height topographs of uncomplexed pBR322 (A) and linear DNA (C) alongside with complexes of these formed when mixed with the chitosan C(0.01,162) (B and D, respectively). cDNA ) 4 µg/mL and k ) 1.

abundance of these. A lower limit of ∼150 bp has been suggested for the DNA to be able to collapse into discrete particles of well-defined morphology, whereas a minimum length of approximately 400 bp has been proposed as a requirement for the formation of a toroidal morphology.23,50 If some of the DNA fragments are shorter than the minimum length required for toroidal formation, the fraction of rods would apparently be larger when compared to toroids, as was observed.1,51 The wider height and contour length distributions observed for the complexes of the linear DNA compared to the plasmid could therefore reflect DNA species within the chain length regions where these changes occur. Because of this and the higher relevance of the plasmid DNA as a vector for gene delivery, the detailed comparison of the influence of chitosan molecular parameters on the complexation was performed using pBR322. No obvious differences were observed in the size and morphologies of the DNA-chitosan structures in the aliquots extracted between 1 and 30 min after blending the solutions, indicating that the kinetics of the complexation was fast, and the structure formation completed within the first minute. Long-term stability of the complexes was investigated by observing the complexes after storage at 4 °C for three months. The TM-AFM topographs showed that the same type of structures, with comparable dimensions and distribution of asphericity indices, appeared after about 3 months as after 10 min. The results in the following describe properties of complexes that were prepared for observation by AFM following 10 min incubation of the polyanion-polycation mixture. Since the electrostatically driven interaction between mica and free chitosan52 represents a possible competition with DNA for the polycation, the observed morphologies do not necessarily correspond to those in the bulk at similar concentrations. However, insignificant changes in the relative abundance, and dimension, of the toroid and rodlike species



Figure 2. Image galleries of toroidal and rodlike ensembles. The chitosans C(0.01,162) (A) and C(0.15,196) (B) were used to form complexes with pBR322. cDNA ) 4 µg/mL and cchit was adjusted to yield k ) 1. Structures were included in the linear ensemble provided A > 0.5 while structures for the toroidal ensemble was restricted to an A ∈ (0.2-0.35) with the additional criteria of the presence of a distinct hole in the center.

Figure 3. Distribution of asphericity indices calculated on the basis of several AFM topographs for DNA-chitosan complexes formed from mixing pBR322 with the chitosans C(0.01,162) (A), C(0.15,196) (B), C(0.35,182) (C), and C(0.49,102) (D). N is the total number of complexes making up the distributions.

was seen when varying k in the interval 0.6-1.2, indicating that this is not a prominent factor here. Effect of the Charge Density of Chitosan on the Morphology of the DNA-Chitosan Complexes. Ensembles of DNA-chitosan complexes, consisting of approximately 100 species collected from several AFM topographs, were used for the quantitative analysis of the structures. The image galleries in Figure 2 show the rodlike and toroidal ensembles of the pBR322 plasmid complexed with the C(0.01,162) and

C(0.15,196) chitosans when k ) 1. The fraction of structures included in the analysis was for all of these chitosans at least 90%. Distributions of asphericity indices for complexes formed by the four different high DP chitosans with varying FA (C(0.01,162), C(0.15,196), C(0.35,182), and C(0.49,102)) and at k ) 1 (Figure 3) all show a peak centered at the ideal value of a toroid (A ) 0.25). The distribution in the region A > 0.5 depended on the chitosan and contained a peak around A ) 0.8 for the chitosans with FA ) 0.15, 0.35, and

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Danielsen et al. Table 2. Contour Lengths of the Toroidal and Rodlike Complexes Formed from Linear and Plasmid DNA When Mixed with the Chitosans C(0.01,162), C(0.15,196), C(0.35,182), and C(0.49,102)

Figure 4. Ratio between the fraction of toroids and rods as a function of FA, when the high DP chitosans was used in the complex formation and k ) 1. The DNA used was the pBR322 with a concentration of 4 µg/mL.

0.49 (Figure 3B-D). This peak was completely lacking for FA ) 0.01 (Figure 3A), reflecting an absence of rodlike structures for DNA polyplexes with the chitosan C(0.01,162). The distinction between toroids and globules, both having A < 0.5, was, however, not as straightforward and was additionally guided by visual inspection. The reason for this is most probably due to the convolution of the AFM tip with the structures in the capturing process, yielding globular species that do not appear as perfect spheres and thus have A > 0. Polyplexes were included in the toroidal ensemble provided A ∈ (0.2-0.35) and using the additional criteria of the presence of a distinct hole in the center of the structure, whereas the globular ensemble consisted of species devoid of the hole. Thus, the peak centered at A ) 0.25 in the distributions contains both the toroidal and the globular structures and does not necessarily reflect the true toroid content in the population. The focus on the formation of toroids is prominent in the literature on DNA complexation and condensation,16,49,53 but as our results show, compaction of DNA with chitosan yields additionally a large fraction of rods. Theoretical studies have shown that the chain stiffness is an important factor in determining the morphology of collapsed structures.34,35 Although the toroid is the most stable morphology for a rather stiff chain, the rod is the most stable one when the chain stiffness is decreased.35 Our results therefore indicate that the chain stiffness of DNA (persistence length Lp ) 50 nm) is within the range where a coexistence of the rodlike and toroidal morphologies may occur for the intersegment attraction mediated by the complexation using chitosan. The toroid-to-rod ratios for the DNA-chitosan complexes (k ) 1) were found to decrease with decreasing charge density of the chitosan used (Figure 4). Since the intersegment attraction within the complexes is expected to be strongly dependent on the total charge of the polycation, these data indicate that the toroidal shape is preferred for strong intersegment attractions. Varying the Charge Ratio for the Preparation of the DNA-Chitosan Complexes. The above results were ob-

DNA

chitosan

[qchit]/ [qDNA]

rods 〈Lc〉 ( sd (nm)

toroids 〈Lc〉 ( sd (nm)

linear plasmid plasmid plasmid plasmid plasmid plasmid

C(0.01,162) C(0.01,32,) C(0.01,162) C(0.01,162) C(0.15,196) C(0.35,182) C(0.49,102)

1 1 1 6 1 1 1

138 ( 66 (N ) 21) 135 ( 50 (N ) 17) 139 ( 56 (N ) 17) 183 ( 92 (N ) 32) 125 ( 37 (N ) 74) 128 ( 37 (N ) 100) 113 ( 32 (N ) 92)

120 ( 45 (N ) 34) 119 ( 13 (N ) 8) 128 ( 76 (N ) 38) 148 ( 35 (N ) 30) 120 ( 35 (N ) 59) 123 ( 28 (N ) 72) 104 ( 26 (N ) 22)

tained employing a charge ratio of chitosan to DNA (k) equal to one. The surface charge of the complexes is expected to influence their interaction with blood components and the negatively charged cell membrane, and an increased transfection for DNA-polycation complexes using excessive amounts of the polycation has been reported.9,11,54 Additionally, positively charged chitosan-based nanoparticles have been reported to result in lower gene expression and immune responses.55 Previous studies using either chitosan or polyL-lysine indicate that the size and stability of the polyplexes may vary with the charge ratios within the polyplexes.12,20,29,32,33 These observations stimulated studies with k larger than one, while maintaining constant cDNA. Our results showed that when k was increased from 1 to 6 using the chitosan C(0.01,162), the toroid-to-rod ratio decreased. In addition to the well-defined toroids and rods, also other structures that were not seen for k ) 1 were observed at k ) 6. These were racquet-like, flowerlike, or pretzel-like shapes, similar to those identified as kinetically trapped xanthan-chitosan polyplexes.43 A possible scenario is that the large excess of chitosan present when the complex formation takes place leads to an increased number of contact points between DNA segments and the chitosan, both within and between single DNA molecules. Such numerous interactions between chitosan and DNA might result in the complexes being trapped in intermediate states.3 Increasing k from 1 to 6 further resulted in a decrease in the width of the height distribution from 12 to 5 nm. The width of the height distribution is here defined as the height interval between the 5% and 95% percentile of the cumulative height distribution. In addition to getting narrower, the main peak in the height distribution of the toroidal species was observed to be shifted toward smaller heights, whereas the additional peaks in the height distribution for the rodlike ensemble disappeared when the amount of chitosan was increased. Since no obvious change in the contour length was observed upon increasing k (Table 2), the decrease in the height might indicate a more compact arrangement of the DNA within the complex56 or that the complex consists of fewer DNA molecules. Influence of the Degree of Polymerization of the Polycation on the Morphology of the Polyplexes. The interaction of a charged ligand with DNA and the transfection efficiency of the polyplexes are reported to be strongly dependent on the valence of the ligand.29,57 Recent transfection studies, both in vitro in different cell lines (293 cells28,29 and EPC cells58) and in vivo in mouse lungs (by measuring



Figure 5. Tapping mode AFM topographs showing the compacted structures resulting from mixing pBR322 with the low DP chitosan C(0.15,4) at k ) 1 (A) and k ) 6 (B). cDNA ) 4 µg/mL.

luciferase expression after intratracheal administration),29 suggest that employing low molecular weight chitosans for the DNA complexation increase the transfection while at the same time maintain their ability to form complexes with DNA. An additional facet is the reported reduced cytotoxicity connected to a reduced valence of the polycations as reported for, among others, PLL,57 PEI,59 and chitosan.14 When the molecular weight of the chitosans was decreased from ∼200 kDa to ∼10 kDa, while keeping k ) 1, the typical toroidal and rodlike structures disappeared and were replaced by less well-defined structures. The lack of toroidal and rodlike species for pBR322-C(0.15,4) polyplexes (Figure 5A) was also observed when the chitosans C(0.35,12) and (0.49,22) were used. Clusters of both free DNAs bundled together and several small globules sticking together were observed (Figure 5A). The large amounts of DNA segments in the clusters clearly indicate that the chitosans are not capable of fully condensing the DNA at these conditions. As k was increased 6-fold, the toroidal and rodlike structures were again dominating (Figure 5B). This reduced ability of low DP chitosans to condense DNA compared to the chitosans with higher DP indicates that they have a lower affinity for binding to DNA. The binding constant between a ligand and a polymer has been reported to be strongly dependent on the valence of the ligand, with a low valence yielding a weak binding.60-62 When the DP of the chitosan is reduced, the resulting reduction of the valence is expected to decrease its affinity to DNA. The cchit/cDNA ratio required to complete the complexation process therefore depends on the valence, and thus the DP, of the chitosan. The disappearance of rods when lowering the DP is also seen directly from the distribution of asphericity indices (Figure 6), as the number of species with A > 0.5 are largely reduced for the complexes made from C(0.15,4). Increasing k to 6 for the low DP chitosan C(0.15,4) resulted in a toroid-to-rod ratio comparable to that of the high DP chitosans at k ) 1. No obvious differences were observed in the dimensions of the polyplexes when high or low DP chitosans (C(0.01,32), C(0.15,4), C(0.01,162), and C(0.15,196)) were used. The major factors in determining the size of the toroidal and rodlike DNA condensates therefore appear to be inherent properties of the DNA molecule. Dimensions of DNA-Chitosan Complexes. The average contour length 〈Lc〉 of the toroidal and rodlike DNA-chitosan polyplexes was found to be about 130 nm, independent of the linear or toroidal geometry of the complex and the

Figure 6. Distributions of asphericity indices for DNA-chitosan complexes formed from pBR322 and the chitosans C(0.15,4) at k ) 1 (A) and 6 (B), and C(0.15,196) at k ) 1 (C). cDNA ) 4 µg/mL.

chitosan employed (Table 2). This is less than 1/10 of the contour length of the uncomplexed plasmid of 1.5 µm. The distributions of the contour lengths of the complexes showed a standard deviation in the order of 20-40% of the mean value (Table 2). In previous studies, contour length measurements have been used to predict the folding pathway of the DNA when complexed with PLL. A contour length of the rods measured to be about half the contour length of the toroids was taken as a support for the suggestion that some toroids may form from rods that open into toroids45,46 or that toroids collapse to form rods.33 Our results revealed no differences between the contour lengths of toroids and rods and, thus, indicate that none of the structures seem to be a precursor of the other.3 The height distribution histograms (Figure 7) of the DNAchitosan complexes (k ) 1) formed from pBR322 and the chitosans C(0.01,162), C(0.15,196), C(0.35,182), and C(0.49,102) show that all rodlike complexes reveal a main peak close to 4 nm. For comparison, the height of the DNA was determined to be 0.3 nm, which coincides with other reports using AFM.15,45,63 The height distributions were similar for the various chitosans, except for FA ) 0.01 where several additional peaks and a tail extending up to 26 nm were observed (Figure 7). The shape of the height distributions for the toroidal complexes was, except for FA ) 0.01, similar to that of the rods, but with the main peak slightly

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Figure 7. Normalized height distributions for the rodlike and toroidal DNA-chitosan complexes when pBR322 was complexed with the four different chitosans C(0.01,162), C(0.15,196), C(0.35,182), and C(0.49,102). cDNA ) 4 µg/mL and cchit was adjusted to yield k ) 1.

shifted toward larger heights. The additional peaks appearing, mainly for the linear complexes, indicate that the complexes that have more than one association mode consist of different numbers of DNA chains or combinations of these possibilities. The apparent noisy distributions obtained for FA ) 0.01 could arise from the higher charge density of this chitosan, resulting in stronger interactions and increased possibility of being kinetically trapped. Wrapping one single plasmid DNA into a toroid with the observed mean circumference would yield a cross-section of about 10-12 DNA double-strands packed close together. This corresponds to a height of approximately 7-10 nm, assuming hexagonal packing of the DNA strands with an interhelical spacing of 2.8 nm.49,56 Taking into consideration that the AFM tip may compress the complexes during capturing, this indicates that the DNA-chitosan complexes observed here consist of one or a few DNA molecules. Covariation between the mean height and contour length of the toroidal and rodlike complexes (Figure 8) show that several different mean heights can occur for the same contour length of the polyplexes. This indicates that different complexes may involve different numbers of DNA molecules. In a model where the plasmid is wrapped into a toroid with increasing number of turns, or similarly in a rodlike overall geometry, one would expect a decrease in the mean height of the complex with increasing length of the complex. Although there is a vague indication of this trend in the data for the complexes prepared using the chitosan C(0.15,196), the data is not sufficient to be conclusive on this point. The height versus distance determined continuously along the complexes further revealed considerable height variations within some complexes, indicating a coexistence of different packing modes also within a complex. The presence of only a few DNA molecules in each compacted structure is consistent with Golan et al.,45 who suggested that most

condensed DNA complexes contain a single DNA molecule, whereas a few complexes contain two or three DNA molecules. Thus, the results obtained here do not support the suggestions that multiple DNA molecules necessarily are required to achieve condensation when the DNA molecule is shorter than 40 kbp.50 Both the dimensions64 and the packing density50 of the DNA condensates have been suggested to be constants. However, if only very few DNA molecules are involved in the complexation, at least one of the above parameters must vary. The size and shape of the globular polyplexes were found to depend on the employed chitosan. In general, the observed globular structures can be divided into two subclasses. The first class contains globular structures with a size of the same order, or larger, as the toroids and was prominent when using the chitosans C(0.01,162) and C(0.01,32). The other class of globular structures consists of very small globules with a hmax ∼ 7 nm and a volume of the same order as that of one single pBR322 DNA molecule (∼ 4500 nm3) and was prominent for complexes formed from the chitosan C(0.49,102). The relative amount of these species was found to increase with increasing FA of the chitosan when high DP chitosans were used, and further observed as parts of larger aggregates. The globular structures obtained using C(0.01,162) were higher and had a broader distribution, compared to those obtained with C(0.49,102). The appearance of the various structures achieved for the different chitosans can be explained by the different nature of the binding to DNA. The binding of a ligand to a polymer is described to be anti-cooperative with respect to the influence of a bound ligand on the binding affinity of the next interacting ligand to the same polymer.5 The nature of the binding is also shown to depend on the ionic strength (I).17 Studying the properties of PLL-DNA complexes, Liu et al.17 observed that the binding of PLL to DNA was



Figure 8. Mean height of individual rodlike (A,C) and toroidal (B,D) complexes plotted against its contour length. Plots are shown for pBR322 complexed with the chitosans C(0.15,196) (A,B) and C(0.35,182) (C,D). cDNA ) 4 µg/mL with a cchit yielding k ) 1.

anticooperative at low I, but became cooperative when I was increased, with the crossover depending on the chain length of PLL. While multimolecular toroids and rods were prominent for anticooperative binding, condensation of a single DNA molecule into small spheroids or ellipsoidal rods occurred for cooperative binding. Based on these ideas the different structures observed here is proposed to be due to variations in the valence of the chitosan, affecting the nature of the binding within the polyplex. The small globules observed for the C(0.49,102)-DNA complexes indicate cooperative binding of the chitosan to DNA, whereas the high charge density chitosan C(0.01,162) binds in an anticooperative way to form toroids, rods, and larger globules. The coexistence of small globules, toroids, and rods for some of the chitosans indicates that the complexation has taken place close to the crossover point between cooperative and anticooperative binding. Furthermore, the different structures formed when using the two chitosans C(0.01,32) and C(0.49,102), having comparable total valence, but different charge density, show that both the structure and the valence of the polycation are important factors in determining the resulting structures of the DNA condensates. Conclusion This study demonstrates that chitosan effectively condense DNA into toroidal, rodlike and globular structures with the dominating topology depending on both the FA and the DP of the chitosan. Decreasing the charge density of the chitosan resulted in a decrease in the toroid-to-rod ratio, indicating that the strength of the intersegment interaction, mediated by the charge of the chitosan, is important in determining the shape of the DNA-chitosan complexes with the toroid

being the preferred structure for high intersegment attractions. High and low DP chitosans yielded the same type of structures with comparable dimensions. However, to compensate for the reduced interaction strength of the low DP chitosans with DNA, an increased concentration of the low DP chitosans was needed compared to the high DP chitosans, to be able to form these well-defined structures. Measuring the dimensions of the toroidal and rodlike complexes showed that, in contrast to their relative abundance, neither the height nor the contour length was affected by the chitosan molecular parameters. Work in progress includes chitosan samples selected from optimalization of the transfection and studies of the polyplex stability when challenged by other polyanions. Acknowledgment. This work is supported by The Norwegian Research Council (Grant Nos. 129104/420 and 121894/420). References and Notes (1) Arscott, P. G.; Li, A. Z.; Bloomfield, V. A. Biopolymers 1990, 30, 619-630. (2) Chattoraj, D. K.; Gosule, L. C.; Schellman, J. A. J. Mol. Biol. 1978, 121, 327-337. (3) Bloomfield, V. A. Biopolymers 1997, 44, 269-282. (4) Manning, G. S. Q. ReV. Biophys. 1977, 11, 179-246. (5) Rouzina, I.; Bloomfield, V. A. Biophys. Chem. 1997, 64, 139-155. (6) Matulis, D.; Rouzina, I.; Bloomfield, V. A. J. Mol. Biol. 2000, 296, 1053-1063. (7) Wilson, R. W.; Bloomfield, V. A. Biochemistry 1979, 18, 2196 (8) Plum, G. E.; Arscott, P. G.; Bloomfield, V. A. Biopolymers 1990, 30, 631-643. (9) Jones, N. A.; Hill, I. R. C.; Stolnik, S.; Bignotti, F.; Davis, S. S.; Garnett, M. C. Biochim. Biophys. Acta 2000, 1517, 1-18. (10) Howard, K. A.; Dash, P. R.; Read, M. L.; Ward, K.; Tomkins, L. M.; Nazarova, O.; Ulbrich, K.; Seymour, L. W. Biochim. Biophys. Acta 2000, 1475, 245-255.

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Structural Analysis of Chitosan Mediated DNA Condensation by AFM

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