Bundling and Aggregation of DNA by Cationic Dendrimers - American

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Bundling and Aggregation of DNA by Cationic Dendrimers E. Froehlich,† J. S. Mandeville,† C. M. Weinert,‡ L. Kreplak,‡ and H. A. Tajmir-Riahi*,† Department of Chemistry-Biology, University of Que´bec at Trois-Rivie`res, C.P. 500, Trois-Rivie`res (Que´bec), Canada G9A 5H7, and Department of Physics, Sir James Dunn Building, Dalhousie University Lord Dalhousie Drive, Halifax, Canada NS B3H 3J5 Received November 3, 2010; Revised Manuscript Received December 2, 2010

Dendrimers are unique synthetic macromolecules of nanometer dimensions with a highly branched structure and globular shape. Among dendrimers, polyamidoamine (PAMAM) have received most attention as potential transfection agents for gene delivery, because these macromolecules bind DNA at physiological pH. The aim of this study was to examine the interaction of calf-thymus DNA with several dendrimers of different compositions, such as mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) at physiological conditions, using constant DNA concentration and various dendrimer contents. FTIR, UV-visible, and CD spectroscopic methods, as well as atomic force microscopy (AFM), were used to analyze the macromolecule binding mode, the binding constant, and the effects of dendrimer complexation on DNA stability, aggregation, condensation, and conformation. Structural analysis showed a strong dendrimer-DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of KmPEG-G3 ) 1.5 ((0.5) × 103 M-1, KmPEG-G4 ) 3.4 ((0.80) × 103 M-1, and KPAMAM-G4 ) 8.2 ((0.90) × 104 M-1. The order of stability of polymer-DNA complexation is PAMAM-G4 > mPEG-G4 > mPEG-G3. Both hydrophilic and hydrophobic interactions were observed for dendrimer-DNA complexes. DNA remained in the B-family structure, while biopolymer particle formation and condensation occurred at high dendrimer concentrations.

Introduction Dendrimers form stable complexes with DNA and protect DNA against degradation by nucleases.1-5 Such properties make dendrimers excellent tools for gene delivery.5-8 Formation of a complex between DNA and a synthetic carrier appears to be the critical parameter for nonviral gene delivery. A number of synthetic vectors including polylysines, cationic liposomes, and cationic polymers have been used for transfer of DNA into cells.9-12 Among dendrimers, polyamidoamine (PAMAM; Figure 1A) forms complexes with nucleic acids on the basis of ionic interaction between negatively charged backbone phosphate group and positively charged amino group (protonated) of polymers at physiological conditions. Short DNA sequences can wrap around dendrimers and allow genetic materials to be delivered to mammalian cells.8 Dendrimers can also mimic biological macromolecules such as enzymes, viral protein, antibodies, histones, and polyamines such as spermine and spermidine.13-15 However, PAMAM dendrimers are toxic in cells and animals due to their polycationic character.16 It has been demonstrated that modification of the amino groups on the periphery of the dendrimer with poly(ethylene glycol) chains reduces the toxicity and increases the biocompatibility of the resulting polymer (Figure 1B,C).2,17,18 This is because poly(ethylene glycol) is nontoxic, nonimmunogenic, and watersoluble, and its conjugation with other substrates produces conjugates that combine the properties of both the substrate and the polymer. However, conjugate formation can alter the binding affinity of PAMAM to DNA, drug, and protein in general. Among polycationic agents, dendrimers are known to induce DNA condensation effectively and produce different types of * To whom correspondence should be addressed. Tel.: 819-376-5011 (ext. 3310). Fax: 819-376-5084. E-mail: [email protected]. † University of Que´bec at Trois-Rivie`res. ‡ Dalhousie University Lord Dalhousie Drive.

particles with DNA wrapping around dendrimers.19 We have recently reported DNA condensation and the conformational transitions induced by cationic lipids, using spectroscopic methods and atomic force microscopy (AFM).20 Therefore, it was of interest to use spectroscopic studies in combination with AFM imaging in order to analyze the nature of dendrimer-DNA interaction and to establish the correlation between the spectroscopic data and the structural diversity of polymer-DNA complexes. We report the interaction of calf-thymus DNA with mPEGPAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) (Figure 1; mPEG, methoxypoly(ethylene glycol)) at physiological conditions, using constant DNA concentration and various polymer contents. Fourier transform infrared (FTIR), circular dichroism (CD), and UV-visible spectroscopic methods and atomic force microscopy (AFM) were used to measure the dendrimer binding site, binding constant, DNA aggregation, condensation, and conformation in the polymer-DNA complexes. Our spectroscopic study provides a major structural analysis of dendrimer-DNA interaction, which helps elucidate the nature of this biologically important complexation in vitro.

Experimental Section Materials. Highly polymerized type I calf-thymus DNA sodium salt (7% Na content) was purchased from Sigma Chemical Co., and was deproteinated by the addition of CHCl3 and isoamyl alcohol in NaCl solution. To check the protein content of DNA solution, the absorbance at 260 and 280 nm was recorded. The A260/A280 ratio was 1.85, showing that the DNA was sufficiently free from protein.21 PAMAM-G4 (MW 14214 g/mol) was purchased form Aldrich Chemical Co and used as supplied. m-PEG-PAMAM-G3 (MW 5697 g/mol) and m-PEG-PAMAMG4 (MW 8423 g/mol) were synthesized according to published methods.22,23 Other chemicals were of reagent grade and used without further purification.

10.1021/bm1013102  2011 American Chemical Society Published on Web 12/30/2010

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Froehlich et al. DNA solution was calculated to be 25 mM in DNA phosphate. The average length of the DNA molecules estimated by gel electrophoresis was 9000 base pairs (molecular weight ∼6 × 106 Da). The appropriate amount of dendrimer (0.25-1 mM) was prepared in Tris-HCl (pH 7.2). The polymer solution was then added dropwise to DNA solution to attain desired dendrimer content of 0.125, 0.25, and 0.5 mM with a final DNA concentration of 12.5 mM (P) for infrared spectroscopic measurements. FTIR Spectroscopy. Infrared spectra were recorded on a FTIR spectrometer (Impact 420 model), equipped with DTGS (deuterated triglycine sulfate) detector and KBr beam splitter, using AgBr windows. Spectra were collected after 2 h incubation of dendrimer with DNA solution and measured in triplicate. Interferograms were accumulated over the spectral range 4000-400 cm-1 with a nominal resolution of 2 cm-1 and a minimum of 100 scans. The dendrimer concentrations used in infrared were 0.125, 0.25, and 0.5 mM with final DNA content of 12.5 mM. The water subtraction was carried out using distilled water as a reference at pH 7.3.26 A good water subtraction was achieved as shown by a flat baseline around 2200 cm-1 where the water combination mode is located. This method is a rough estimate but removes the water content in a satisfactory way. The difference spectra [(DNA solution + dendrimer) - (DNA solution)] were obtained, using the sharp DNA band at 968 cm-1 as internal reference. This band, which is due to deoxyribose C-C stretching vibration, exhibits no spectral changes (shifting or intensity variation) upon polymer-DNA complexation and is canceled out upon spectral subtraction. The spectra are smoothed with Savitzky-Golay procedure.26 The plots of the relative intensity (R) of several peaks of DNA inplane vibrations related to A-T, G-C base pairs, and the PO2- stretching vibrations such as 1710 (guanine (G)), 1661 (thymine (T)), 1610 (adenine (A)), 1491 (cytosine (C)), and 1225 cm-1 (PO2- asymmetric) versus dendrimer concentrations were obtained after peak normalization using

Ri )

Figure 1. Chemical structures of dendrimers (A) PAMAM-G4, (B) mPEG-PAMAM-G3, and (C) mPEG-PAMAM-G4.

Preparation of Stock Solution. Sodium-DNA was dissolved to 1% w/w (10 mg/mL) in 10 mL of Tris-HCl (pH 7.3) at 5 °C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of the stock calf-thymus DNA solution was determined spectrophotometrically at 260 nm by using molar extinction coefficient of 6600 cm-1 M-1 (expressed as molarity of phosphate groups).24,25 The UV absorbance at 260 nm of a diluted solution (40 µM) of calf-thymus DNA used in our experiments was measured to be 0.25 (path length was 1 cm) and the final concentration of the stock

Ii I968

where Ii is the intensity of absorption peak for pure DNA and DNA in the complex with i concentration of polymer and I968 is the intensity of the 968 cm-1 peak (internal reference).27 CD Spectroscopy. Spectra of DNA and dendrimer-DNA adducts were recorded at pH 7.3 with a Jasco J-720 spectropolarimeter. For measurements in the far-UV region (200-320 nm), a quartz cell with a path length of 0.01 cm was used. Six scans were accumulated at a scan speed of 50 nm per min, with data being collected at every nm from 200 to 320 nm. Sample temperature was maintained at 25 °C using a Neslab RTE-111 circulating water bath connected to the waterjacketed quartz cuvette. Spectra were corrected for buffer signal, and conversion to the Mol CD (∆ε) was performed with the Jasco Standard Analysis software. The dendrimer concentrations used were 0.125, 0.25, 0.5, and 1 mM, with final DNA content of 2.5 mM. Absorption Spectroscopy. The absorption spectra were recorded on a Perkin-Elmer Lambda 40 spectrophotometer with a slit of 2 nm and scan speed of 240 nm min-1. Quartz cuvettes of 1 cm were used. The absorbance assessments were performed at pH 7.3 by keeping the concentration of DNA constant (125 µM), while varying the concentration of dendrimer (5-100 µM). The binding constants of dendrimerDNA complexes were calculated as reported.28,29 It is assumed that the interaction between the ligand L and the substrate S is 1:1, and for this reason, a single complex SL (1:1) is formed. It was also assumed that the sites (and all the binding sites) are independent, and finally, the Beer’s law is followed by all species. A wavelength is selected at which the molar absorptivities εS (molar absorptivity of the substrate) and ε11 (molar absorptivity of the complex) are different. Then at total concentration St of the substrate, in the absence of ligand and the light path length is b ) 1 cm, the solution absorbance is

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Ao ) εSbSt

513

(1)

In the presence of ligand at total concentration Lt, the absorbance of a solution containing the same total substrate concentration is

AL ) εSb[S] + εLb[L] + ε11b[SL]

(2)

(where [S] is the concentration of the uncomplexed substrate, [L] is the concentration of the uncomplexed ligand, and [SL] is the concentration of the complex) which, combined with the mass balance on S and L, gives

AL ) εSbSt + εLbLt + ∆ε11b[SL]

(3)

where ∆ε11 ) ε11 - εS - εL (εL molar absorptivity of the ligand). By measuring the solution absorbance against a reference containing ligand at the same total concentration Lt, the measured absorbance becomes

A ) εSbSt + ∆ε11b[SL]

(4)

Combining eq 4 with the stability constant definition K11 ) [SL]/[S][L] gives

∆A ) K11∆ε11b[S][L]

(5)

where ∆A ) A - Ao. From the mass balance expression St ) [S] + [SL], we get [S] ) St/(1 + K11[L]), which is eq 5, giving eq 6 at the relationship between the observed absorbance change per centimeter and the system variables and parameters.

StK11∆ε11[L] ∆Α ) b 1 + K11[L]

(6)

Equation 6 is the binding isotherm, which shows the hyperbolic dependence on free ligand concentration. The double-reciprocal form of plotting the rectangular hyperbola 1/y ) f/d × 1/x + e/d, is based on the linearization of eq 6 according to the following equation:

1 1 b ) + ∆A StK11∆ε11[L] St∆ε11

(7)

Thus, the double reciprocal plot of 1/∆A versus 1/[L] is linear and the binding constant can be estimated from the following equation:

K11 )

intercept slope

(8)

Atomic Force Microscopy. Dendrimer-DNA complexes at a ratio of 1:1 and final DNA concentration of 0.1 mM were prepared in 5 mL tris-HCl (pH 7.4). The solutions were either used undiluted or diluted further in ultrapure water. For each sample, a 30 µL aliquot was adsorbed for two minutes on freshly cleaved muscovite mica. The surface was rinsed thoroughly with 10 mL of ultrapure water and dried with argon. AFM imaging was performed in acoustic mode at a scanning speed of 1 Hz with an Agilent 5500 (Agilent, Santa Barbara, CA) using high frequency (300 kHz) silicon cantilevers with a tip radius of 2-5 nm (TESP-SS, Veeco, Santa Barbara, CA). Images were treated using the software Gwyddion (http://gwyddion.net/).

Figure 2. FTIR spectra and difference spectra [(DNA solution + dendrimer solution) - (DNA solution)] in the region of 1800-600 cm-1 for the free calf-thymus DNA and free mPEG-PAMAM-G3 (A), free mPEG-PAMAM-G4 (B), and free PAMAM-G4 (C) and their complexes in aqueous solution at pH 7.3 with various polymer concentrations (0.125 and 0.5 mM) and constant DNA content (12.5 mM).

Results FTIR Spectra of Dendrimer-DNA Complexes. The IR spectral features for dendrimer-DNA interaction are presented in Figures 2 and 3. The assignments of DNA main vibrational frequencies are given in Table 1.30-34 Dendrimer-Phosphate Binding. Strong polymer-PO2 interaction is evident from increase in intensity and shifting of the PO2 asymmetric band at 1225 and symmetric band at 1088 cm-1, in the spectra of the polymer-DNA complexes (Figures 2 and 3A-C). The PO2 band at 1225 cm-1 gained intensity and shifted toward a higher frequencies at 1234 (mPEGPAMAM-G3), 1237 (mPEG-PAMAM-G4), and 1232 (PAMAMG4), while the band at 1088 gained intensity in the spectra of mPEG-PAMAM-G3, mPEG-PAMAM-G4 and PAMAM-G4DNA complexes (Figures 2 and 3A-C, complexes with 0.5 mM). The positive features at 1238-1227 cm-1 (PO2 antisymmetric stretch) in the difference spectra of mPEG-PAMAMG3-, mPEG-PAMAM-G-, and PAMAM-G4-DNA complexes are due to increase in intensity of the phosphate vibrational frequencies upon dendrimer interaction (Figure 2A-C diffs). Further evidence regarding strong polymer-PO2 interaction is also coming from the intensity ratio variations of symmetric and asymmetric PO2 bands at 1088/1222 due to ligand-PO2 interaction.26 The ratio of νs/νas was changed from 1.40 (free DNA) to 2.35 (mPEG-PAMAM-G3-DNA), 2.50 (mPEGPAMAM-G4-DNA), and 3.1 (PAMAM-DNA), upon polymer complexation (Figure 2A-C). The extent of the PO2 intensity ratios variations indicates a stronger polymer-PO2 interaction for PAMAM than those of the mPEG-PAMAM-G3 and mPEGPAMAM-G4 complexes. Similar spectral changes were observed for the backbone PO2 stretching vibrations in the IR spectra of cationic lipid-DNA complexes, where strong lipid-phosphate binding occurred.20

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Froehlich et al. Table 1. Principal Infrared Absorption Bands, Relative Intensities, and Assignments for Calf-Thymus DNA in Aqueous Solution at pH 7 ( 0.2 wavenumber (cm-1) intensitya 1710 1661 1610 1579 1527 1491 1225 1088 1053 968 892 834

vs vs s sh w m vs vs s s m m

assignment

30-34

guanine (CdO stretching) thymine (C2dO stretching) adenine (C7dN stretching) purine stretching (N7) in-plane vibration of cytosine and guanine in-plane vibration of cytosine asymmetric PO2- stretch symmetric PO2- stretch C-O deoxyribose stretch C-C deoxyribose stretch deoxyribose, B-marker deoxyribose-phosphate, B-marker

a Intensities: s ) strong, sh ) shoulder, vs ) very strong, m ) medium, w ) weak.

Figure 3. Intensity ratio variations for DNA infrared in-plane vibrations upon dendrimer complexation for (A) mPEG-PAMAM-G3-DNA, (B) mPEG-PAMAM-G4-DNA, and (C) PAMAM-G4-DNA.

Dendrimer-Base Binding. Evidence for dendrimer-base binding comes from the spectral changes observed for both free DNA and free dendrimer upon complexation. At low polymer concentration 0.125 mM, a major shifting of the bands at 1710 (guanine), 1661 (thymine), and 1610 cm-1 (adenine) toward lower frequency was observed. The shifting was accompanied by a minor increase of intensity for the bands at 1710 (guanine), 1661 (thymine), and 1610 cm-1 (adenine) for the dendrimer-

DNA complexes (Figure 3A-C, 0125 mM). The increase in intensity of these vibrations was characterized by the presence of several positive features at 1710-1702 (guanine), 1665-1650 (thymine), and 1608-1603 cm-1 (adenine) in the difference spectra of mPEG-PAMAM-G3, mPEG-PAMAM-G4, and PAMA-G4-DNA complexes (Figure 2A-C, diff. 0.125 mM). The observed spectral changes are due to major polymer-DNA interaction at low dendrimer concentration. At higher polymer concentrations, the guanine band at 1710 cm-1 shifted to 1709 (mPEG-PAMAM-G3), 1704 (mPEG-PAMA-G4), and 1695 cm-1 (PAMAM-G4), upon DNA complexation (Figure 2A-C, complexes 0.5 mM). The shift of these vibrations was associated with a minor increase to the intensity of the bands 1710 and 1661 cm-1 in the spectra of mPEG-PAMAM-G3 and mPEGPAMAM-G4, while a major reduction of intensity occurred for PAMAM-DNA complexes (Figure 3A-C, diff. 0.5 mM). The increase in the intensity and the shifting of these vibrations is due to polymer-base interaction via guanine N7 and thymine O2 atoms in mPEG-PAMAM-G3 and mPEG-PAMAM-G4 complexes, while the reduction of intensity in the spectra of PAMAM-G4-DNA adducts (with negative feature at 1705 cm-1) is related to major DNA condensation in the presence of PAMAM-G4 at high polymer concentration. This is consistent with our CD spectroscopic results and AFM images that will be discussed further on. The major spectral changes observed for guanine, thymine and adenine bands are indicative of polymer bindings to guanine and adenine N7 reactive sites (major groove) and thymine O2 atom (minor groove), as well as to the backbone phosphate group of DNA duplex. Hydrophobic Interactions. To determine the presence of hydrophobic contact in the dendrimer-DNA complexes, the spectral changes of the polymer antisymmetric and symmetric CH2 stretching vibrations, in the region of 3000-2800 cm-1 were investigated by infrared spectroscopy. The CH2 bands of the free mPEG-PAMAM-G3 located at 2946, 2884, and 2859 cm-1 shifted to 2950, 2883, and 2856 cm-1 (mPEG-PAMAMG3-DNA); free mPEG-PAMAM-G4 with CH2 bands at 2942 and 2876 cm-1 shifted to 2956, 2919, 2884, and 2852 cm-1 (mPEG-PAMAM-G4-DNA) and free PAMAM-G4 with CH2 bands at 2956 and 2856 cm-1 shifted to 2937 and 2950 cm-1 (PAMAM-G4-DNA) in the dendrimer-DNA complexes. The shifting of the polymer antisymmetric and symmetric CH2 stretching vibrations in the region 3000-2800 cm-1 of the infrared spectra suggests the presence of hydrophobic interactions via polymer aliphatic chain and hydrophobic region in DNA.

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Figure 4. CD spectra of calf thymus DNA in Tris-HCl (pH ∼ 7) at 25 °C (2.5 mM) and mPEG-PAMAM-G3 (A), mPEG-PAMAM-G4 (B), and PAMAM-G4 (C) with 125, 250, 500, and 1000 µM polymer concentrations.

CD Spectra and DNA Conformation. The CD spectra of the free DNA and its complexes with different polymer concentrations are shown in Figure 4. The CD of the free DNA composed of four major peaks at 211 (negative), 221 (positive), 245 (negative), and 275 nm (positive; Figure 4). This is consistent with CD spectra of double helical DNA in B conformation.35,36 Upon dendrimer interaction no major shifting of the CD bands was observed at low polymer concentration (0.125-0.25 mM), whereas at higher polymer content (0.5 and 1 mM) a major increase in molar ellipticity of the band at 211 nm and the positivity of the band at 245 was reduced, while the intensity of the band at 275 nm decreased in the spectra of mPEG-PAMAM-G3 and mPEG-PAMAM-G4-DNA complexes (Figure 4A,B). However, the CD band at 275 nm lost its intensity and collapsed in the spectra of PAMAM-G4-DNA complexes at low and high polymer concentrations (Figure 4C). The major loss of intensity of the CD band at 275 nm in the spectra of PAMAM-G4-DNA complexes is due to DNA condensation in the presence of the PAMAM-G4 dendrimer

Figure 5. UV-visible results of calf-thymus DNA and its mPEGPAMAM-G3 (A), mPEG-PAMAM-G4 (B), and PAMAM-G4 (C) complexes: (A) spectra of (a) free DNA (40 µM); (b) free polymer (100 µM); (c-n) mPEG-PAMAM-G3-DNA complexes c (5), d (10), e (15), f (20), g (25), h (30), i (35), j (40), k.(50), l (60), m (70) and n (80 µM). (B) spectra of a) free DNA (40 µM); b) free polymer (100 µM); c-n) mPEG-PAMAM-G4-DNA complexes c (5), d (10), e (15), f (20), g (25), h (30), i (35), j (40), k (50), l (60), m (70), and n (80 µM). (C) Spectra of (a) free DNA (40 µM); (b) free polymer (100 µM); (c-n) PAMAM-G4-DNA complexes c (5), d (10), e (20), f (30), g (40), h (5), i (50), and j (60 µM). Plot of 1/(A - A0) vs (1/polymer concentration) for polymer and calf-thymus DNA complexes, where A0 is the initial absorbance of DNA (260 nm) and A is the recorded absorbance (260 nm) at different polymer concentrations (5-80 µM) with constant DNA concentration of 100 µM at pH 7.4 for mPEG-PAMAM-G3 (A′), mPEG-PAMAM-G4 (B′), and PAMAM-G4 (C′).

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Figure 6. Tapping mode AFM images of DNA in complex with dendrimers. (A) mPEG-PAMAM G3-DNA, in that case we only observed networks of DNA molecules coated and cross-linked by the dendrimers. (B) mPEG-PAMAM G4-DNA, bundles of DNA (arrows) are visible as well as networks. Along the bundles, rings are visible (arrowhead in inset) that we attribute to two “naked” DNA strands that repel each other. An asterisk marks the region blown up in the inset. (C) PAMAM G4-DNA, we observed few free DNA molecules and large flat complexes (solid line) with a dense central core similar to those observed for a mixture of positively charged lipid (DOTAP) and DNA in a previous study.17

(Figure 4C). This is consistent with our AFM images that will be discussed further on. This is also consistent with our infrared results on the dendrimer-DNA complexes that showed no conformational changes for B-DNA with marker bands at 1710 (G), 1225 (PO2), and 834 cm-1 (phosphodiester mode; Figure 2A-C). In a complete B to A transition, the B-DNA marker IR bands are shifted from 1710 to 1700 cm-1 (G), 1225 to 1240 cm-1 (PO2), and 834 to 810 cm-1 (phosphodiester).32,33 The shifting of the guanine bands at 1710 to lower frequency and the PO2 band at 1225 to higher frequency is due to polymer interaction with guanine N7 site and the backbone phosphate group and not due to DNA conformational transition, because the phosphodiester band at 834 cm-1 showed no major shifting in the spectra of dendrimer-DNA complexes (Figure 2A-C). Stability of Dendrimer-DNA Complexes. The dendrimerDNA binding constant was determined as described in Experimental Section (UV-visible spectroscopy). An increasing polymer concentration resulted in an increase in UV light absorption, as can be observed (Figure 5). This is consistent with a reduction of base stacking interaction due to polymer complexation (Figure 5A-C). The double reciprocal plot of 1/(A - A0) versus 1/(polymer concentration) is linear, and the binding constant (K) can be estimated from the ratio of the intercept to the slope (Figure 5A′-C′). A0 is the initial absorbance of the free DNA at 260 nm and A is the recorded absorbance of complexes at different polymer concentrations. The overall binding constant for dendrimer-DNA complexes is estimated to be KmPEG-G3 ) 1.5 ((0.5) × 103 M-1, KmPEG-G4 ) 3.4 ((0.80) × 103 M-1, and KPAMAM-G4 ) 8.2 ((0.90) × 104 M-1 with the order of stability of polymer-DNA complexes being PAMAMG4 > mPEG-PAMAM-G4 > mPEG-PAMAM-G3 (Figure 5A′C′). The binding constant for PAMAM-G4/DNA is expected to be the highest as there are 64 amino groups on the periphery of the dendrimer as compared with 16 on mPEG-PAMAM-G4 and 8 on mPEG-PAMAM-G3 dendrimers. Additionally, the intramolecular hydrogen bonding between mPEG and the PAMAM dendron should result in a weaker interaction in mPEG-PAMAM/DNA complexes. The binding constants estimated are mainly due to the polymer-base binding and not related to the polymer-PO2 interaction, which is largely ionic and can be dissociated easily in aqueous solution. Ultrastructure of Dendrimer-DNA Complexes. The AFM pictures reveal two different types of interactions between the dendrimers and the DNA molecules (Figure 6). The two mPEG terminated dendrimers tend to coat and bundle DNA molecules (Figure 6A,B). In the case of mPEG-PAMAM-G4, we observed ring-like structures along some of the bundles (Figure 6B, inset) that we attributed to two “naked” DNA molecules repelling each other (Figure 6B, inset, arrowhead). In contrast, PAMAM-G4 was able to compact DNA into aggregates exhibiting a central

core surrounded by a flat region (Figure 6C, solid line). These complexes were similar to those observed for a mixture of positively charged lipid (DOTAP) and DNA, in a previous study.20 The condensation of DNA by dendrimers, particularly with PAMAM-G4 (Figure 6C) is consistent with our IR and CD spectroscopic results discussed earlier (Figure 2A-C and Figure 4A-C).

Discussion Nonviral gene delivery systems especially cationic polymers are receiving much attention.37 The generation of particulate systems with a specific shape and size plays a crucial role in the development of modern gene and drug delivery systems. Dendrimers have been explored extensively in this field because their structure is well-defined and can be tailored to specific applications.38,39 Dendrimer-DNA binding is of major importance for gene delivery and transfection. Over the past decade a number of promising synthetic nonviral gene delivery systems have been developed and a profile of their potential advantages and disadvantages has emerged.1,40 The structural analysis of dendrimer-DNA binding is the first step in gene delivery and DNA condensation, which requires further investigations. Several studies have been reported on the nature of dendrimerDNA complexation.7,12,17 However, the structure and stability of the dendrimer-DNA complexes are not well understood. The supermolecular structures formed between DNA and starburst dendrimers were studied by EPR, CD, and UV spectroscopic methods and melting profiles.41 It was shown that, while dendrimer-DNA complexation occurred via ionic interaction, DNA remained in the B-family structure.41 Other studies showed how DNA condensation and particle formation were influenced by different generations of dendrimers.42 Structural polymorphism of dendrimer-DNA complexes was also investigated showing how dendrimer composition, cationic changes, and concentration can effect DNA polymorphism.19 Indirect analytical methods were used to analyze dendrimer-DNA binding with dendrimers of different generations G2 to G7, providing evidence for ionic interaction in polymer-DNA complexation.43 In this study was also shown that DNA wraps around G7 dendimers, while wrapping of DNA does not occur for G2 and G4 dendrimers.43 It was also demonstrated that DNA complexation with polyamidoamine dendrimers (PAMAM) can have major application for transfection.13,44 Our infrared spectroscopic data showed evidence for direct polymer-PO2 interaction for DNA adducts with mPEGPAMAM-G3, mPEG-PAMAM-G4, and PAMAM-G4 dendrimers. Evidence for polymer-phosphate binding comes from major shifting and intensity variations of the PO2 bands at 1225 and 1088 cm-1. Similarly, the polymer base binding was observed

Dendrimer-DNA Interaction

via guanine N7, adenine N7, and thymine O2 atoms. Evidence for this comes from the major shifting and intensity changes of the guanine band at 1710, thymine band at 1661, and adenine band at 1610 cm-1. Hydrophobic contact was also detected in the polymer-DNA complexes based on the infrared spectral shifting observed for the dendrimer CH2 stretching vibrations, in the region of 3000-2800 cm-1. Our CD spectroscopic results showed supramolecular dendrimer-DNA complex formation with DNA remaining in the B-family structure. However, major DNA condensation and particle formation were observed particularly in the case of PAMAM-G4 dendrimer, where a major reduction in the intensity of CD band at 275 nm was observed. DNA condensation and particle formation were evidenced from AFM imaging of the dendrimer complexes, where DNA bundling and aggregation occurred particularly in the case of PAMAM-G4 dendrimer (Figure 6). Stability of dendrimer-DNA complexes showed more stable DNA adducts with PAMAM-G4, than those of pegylated dendimers with the order of PAMAM > mPEGPAMAM-G4 > mPEGPAMAM-G4, indicating the stabilization of dendrimer-DNA complexation by charge neutralization. The binding constant for PAMAMG4/DNA is highest, as there are 64 amino groups on the periphery of the dendrimer compared with 16 on mPEGPAMAM-G4 and 8 on mPEG-PAMAM-G3 dendrimers. Additionally, the intramolecular hydrogen bonding between mPEG and the PAMAM dendron would result in a weaker interaction in mPEG-PAMAM/DNA complexes. In summary, our spectroscopic results in combination with AFM images provide major structural information regarding dendrimer-DNA formulation, which is important in gene delivery and DNA transfection. Acknowledgment. This work is supported by grants from Natural Sciences and Engineering Research Council of Canada (NSERC).

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