Polymeric effects on DNA condensation by cationic polymers observed by atomic force microscopy

Colloids and Surfaces B: Biointerfaces 75 (2010) 230–238

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Polymeric effects on DNA condensation by cationic polymers observed by atomic force microscopy Lei Liu a , Yan-Lian Yang a , Chen Wang a,∗ , Yang Yao b , Yong-Zheng Ma b , Sen Hou b , Xi-Zeng Feng b,∗ a b

National Center for Nanoscience and Technology, Beijing 100190, PR China College of Life Science, Nankai University, Tianjin 300071, PR China

a r t i c l e

i n f o

Article history: Received 17 February 2009 Received in revised form 21 August 2009 Accepted 26 August 2009 Available online 1 September 2009 Keywords: Plasmid DNA DNA condensation Cationic polymers Atomic force microscopy Gene transfection

a b s t r a c t Compaction of DNA by condensing agents can provide insights into DNA assembly processes, which is keenly related to the essence of gene transfection and gene therapy in vivo. In this paper, the morphology of different cationic polymer/DNA complexes was studied by using atomic force microscopy (AFM), which is keen to the mechanism of DNA condensation induced by amine-based cationic block copolymers with poly(poly(ethylene glycol) methyl ether methacrylate). It is found that the structures and dimensions of condensing agent/DNA complexes are sensitively dependent on the condensing agents. The size of DNA aggregates can be affected appreciably by polymers rather than monomers. The amount of nitrogen elements per polymer unit, rather than the molecular weights of polymers, appears to be more effective on the dimension of the condensates. The impact of the copolymer chain structures on the DNA aggregates indicates an effective venue for regulating the dimensions and structures of the DNA condensates, which is beneficial for optimizing delivery systems for gene transfection. © 2009 Elsevier B.V. All rights reserved.

1. Introduction An important aspect of gene therapy is utilizing appropriate carriers for therapeutic genes to efficiently and safely carry the DNA molecules into the nucleus of the target cells. Naked DNA is unable to efficiently cross cellular barriers by passive diffusion due to their large size, strong negative charge, hydrophilicity and susceptibility to nuclease attack. Therefore, it is important to transfer genes into desired cells using the designed delivery system. The compaction of DNA to nanoscale aggregates is a common process for gene transfection. A variety of condensing agents, such as the natural polyamines, spermidine and spermine, have been studied by using electron microscopy (EM), atomic force microscopy (AFM) and light scattering techniques [1–8]. As one of the reported condensing agents, cationic lipids have been tested for in vitro and in vivo transfection of nucleic acids [9–14]. The flexibility in the design of cationic lipid structure and liposome composition, coupled with the diversity of methods for their preparation and in vivo efficiency, have drawn much interest in using cationic lipids for human gene transfer [9]. However, lipid–DNA complexes are unstable in the presence of serum with changes in size, surface charge and lipid composition, restricting their application in vivo. Anita Mann et al. [15] reported that

∗ Corresponding authors. E-mail addresses: [email protected] (C. Wang), [email protected] (X.-Z. Feng). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.08.040

cationic peptides can be considered as attractive alternatives to lipids for non-viral DNA delivery. Stefaan et al. [16] reported that some representative cationic polymers with distinct chemical structures could be used as condensing agents for DNA condensation and factors for influencing the DNA condensation as well. Recently, some cationic polymers have been explored as new candidates for DNA condensing agents [17–20], some of which are demonstrated as effective gene carriers [17]. The essence of the compaction of DNA into nanostructures is in fact the process of self-assemblies of DNA molecules with cationic molecules. Some reports [21–24] suggest that electrostatic interaction is the dominant factor in the process of DNA condensation. However, the process of DNA compaction is affected by other factors, such as hydrophobic properties, ionic intensity, molecular weights of condensing agents. It is therefore plausible to design different structures of condensing agents to induce DNA to condense into nanostructured aggregates, for regulating the dimension and structure of the DNA aggregates. Tertiary amine-based cationic polymers have recently attracted significant attention due to their potential applications as carriers of DNA or oligonucleotides for gene delivery [16]. For example, poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) has been reported to be efficient polycationic condensing agents for non-viral-DNA delivery owing to inherent amine protonation in physiological media [25–27]. The study on the interactions of PDMAEMA with plasmid DNA showed a high reaction rate and easy dissociation of PDMAEMA-based complexes [28]. It has also been proposed that the polymer chain struc-

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tures could have strong effects on the polymer performance [29–31]. In this paper, five compounds: monomer DMAEMA, homopolymer PDMAEMA, PDMAEMA-based triblock copolymers (copolymer I, copolymer II) and diblock copolymer (copolymer III) were used as DNA condensing agents. The structures of DNA aggregates induced by these compounds were directly observed by AFM. The AFM images of DNA condensates revealed that the condensing agents with different nitrogen weight per polymer unit and distinct positions of poly(poly(ethylene glycol) methyl ether methacrylate) (poly-(PEGMA)) in copolymers may affect the dimension and structure of the condensates. Meanwhile, the physical and chemical properties of the DNA aggregates were diverse due to distinct structures of condensing agents. Therefore, designing the polymer chain structures in copolymers could be considered as a possible venue to regulate the structure and dimension of DNA aggregates for gene transfection. The steric contribution of poly-(PEGMA) block may also be beneficial for improving the biocompatibility of polycation polymers in transferring the system from in vitro to in vivo. The gene transfection experiment proves that some condensing agent could transfer genes into cells.

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2.2. Synthesis of PDMAEMA PDMAEMA was prepared by atom transfer radical polymerization (ATRP) in tetrahydrofuran (THF) using ethyl 2bromoisobutyrate (EBiB) as initiator. The structure of PDMAEMA was shown in Fig. 1b. In a typical experiment, monomer DMAEMA (35.6 mmol, 6 mL), CuBr (0.36 mmol, 51.6 mg) and THF (200 mL) were mixed in a round-bottom flask. The flask was degassed with nitrogen for 3 h before being sealed with a rubber septum. Then HMTETA (0.36 mmol, 98 ␮L) was added by a microsyringe. When CuBr was completely dissolved and the solution became transparent, EBiB (0.356 mmol, 52 ␮L) was added. After polymerized at 40 ◦ C for 12 h, the mixture was diluted with THF, passed through a silica gel column using acetone as an eluent and then precipitated in petroleum ether and dried under vacuum for 24 h. 1 H NMR (400 MHz, CDCl3 ): ı2.36 (–N(CH3 )2 ); ı2.76 (–O–CH2 –CH2 –N(CH3 )2 ); ı4.19 (–COO–CH2 –CH2 –N(CH3 )2 ). Gel permeation chromatography (GPC): Mn = 15,700, Mw/Mn = 1.10. 2.3. Synthesis of triblock copolymer poly(PEGMA)-b-PDMAEMA-b-poly(PEGMA) and PDMAEMA-b-poly(PEGMA)-b-PDMAEMA

2. Materials and methods 2.1. Materials 2-(Dimethylamino)-ethyl methacrylate (DMAEMA, Aldrich) in Fig. 1a and poly(ethylene glycol) methyl ether methacrylate (PEGMA, Aldrich) were passed through a basic alumina column and then distilled prior to polymerization. Copper(I) bromide (CuBr, AR) was purified according to method described by Wan et al. [32] 1,1,4,7,10,10-hexamethyl triethylenetetramine (HMTETA, Aldrich, 98%), 2-bromoisobutyryl bromide (BiBB, Aldrich, 98%) and hexanediamine were used as received. All other reagents and solvents were used without further purification unless stated. Plasmid DNA pEGFP-C2 (4.7 kb) was purified from Escherichia coli DH5␣. The concentration of plasmid DNA was measured through spectrophotometric analysis using DU-7 spectrophotometer (Beckman, USA). DNA was kept in TE buffer (10 mmol/L Tris–HCl, 1 mmol/L EDTA, pH = 8.0).

2.3.1. Synthesis of poly(PEGMA)-b-PDMAEMA-b-poly(PEGMA) (copolymer I) 2.3.1.1. Synthesis of the initiator. The bis-initiator (bis(2bromoisobutyryl) hexanediamide) was synthesized as follows: Under a nitrogen atmosphere, a stirred suspension of hexanediamine (1.15 g, 10 mmol) and anhydrous dichloromethane (300 mL) was cooled to 0 ◦ C using an ice bath. The solution of BiBB (1.22 g, 10 mmol) and dichloromethane (10 mL) was then added dropwise. The mixture was subsequently stirred for 12 h at room temperature and the solution turned out to be white emulsion. After filtration, solvent was removed with rotary evaporation. The obtained white powder was dissolved in 20 mL dichloromethane and extracted with saturated aqueous solution of sodium carbonate for three times. The organic layer was dried over anhydrous magnesium sulfate, filtered and the solvent removed under reduced pressure to obtain the desired product bis(2-bromoisobutyryl) hexanediamide (Scheme 1). Yield: 0.82 g (31%). 1 H NMR (400 MHz,

Fig. 1. Chemical structures of condensing agents. The molecular weights of the compounds are shown in (a)–(e). The values of m and n stand for the degree of polymerization.

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Scheme 1. Synthesis of bis-initiator bis(2-bromoisobutyryl) hexanediamide.

CDCl3 ): 3.259 (4H, –NH–CH2 –); ı1.953 (6H, –C(CH3 )2 Br); 1.561 (4H, –NH–CH2 –CH2 –); 1.378 (4H, –NH–CH2 –CH2 –CH2 –). 2.3.1.2. Synthesis of the triblock copolymer poly(PEGMA)-bPDMAEMA-b-poly(PEGMA) (copolymer I). ATRP of triblock copolymer (Fig. 1c) was carried out in solution with bis(2bromoisobutyryl) hexanediamide as the bis-initiator. In a typical experiment, monomer DMAEMA (35.6 mmol, 6 mL), CuBr (0.36 mmol, 51.6 mg) and THF (200 mL) were mixed in a round-bottom flask. The flask was degassed with nitrogen for 3 h before being sealed with a rubber septum. Then HMTETA (0.36 mmol, 98 ␮L) was added by a microsyringe. When CuBr was completely dissolved and solution became transparent, bis(2-bromoisobutyryl) hexanediamide (0.18 mmol, 73.6 mg) was added. After polymerized at 40 ◦ C for 6 h, the second monomer PEGMA (21.6 mmol, 9.5 mL) was added to the solution and polymerized for another 12 h. The mixture was diluted with THF, passed through a silica gel column using acetone as an eluent and then precipitated in petroleum ether and dried under vacuum for 24 h. 1 H NMR (400 MHz, D2 O): ı2.36, ı2.76 (–O–CH2 –CH2 –N(CH3 )2 ); ı4.19 (–COO–CH2 –CH2 –N(CH3 )2 , –COO–CH2 –CH2 –O–); ı3.72 (–O–CH2 –CH2 –O–); ı3.36 (–O–CH3 ). GPC: Mn = 15,500, Mw/Mn = 1.14. According to the ratio of the terminal methyl groups of DMAEMA to that of PEGMA, composition of the triblock copolymer is PEGMA8 -b-DMAEMA50 -b-PEGMA8 . The structure of copolymer I was shown in Fig. 1c. 2.3.2. Synthesis of PDMAEMA-b-poly(PEGMA)-b-PDMAEMA (copolymer II) Triblock copolymer PDMAEMA-b-poly(PEGMA)-b-PDMAEMA was also prepared in THF with bis(2-bromoisobutyryl) hexanediamide as the bis-initiator. The mixture contained bis(2bromoisobutyryl) hexanediamide (0.18 mmol, 73.6 mg), monomer PEGMA (21.6 mmol, 9.5 mL), CuBr (0.36 mmol, 51.6 mg) and HMTETA (0.36 mmol, 98 ␮L). After bubbled with nitrogen through the solution during 3 h to remove oxygen, copolymerization was carried out at 40 ◦ C for 6 h. Then the second monomer DMAEMA (35.6 mmol, 6 mL) was added to the solution and polymerized for another 12 h. The mixture was passed through a silica gel column using acetone and precipitated in petroleum ether and dried under vacuum for 24 h. 1 H NMR (400 MHz, D2 O): ı2.36 (–N(CH3 )2 ); ı2.76 (–O–CH2 –CH2 –N(CH3 )2 ); ı4.19 (–COO–CH2 –CH2 –N(CH3 )2 , –COO–CH2 –CH2 –O–); ı3.72 (–O–CH2 –CH2 –O–); ı3.36 (–O–CH3 ). GPC: Mn = 47,000, Mw/Mn = 1.16. According to the ratio of the terminal methyl groups of DMAEMA to that of PEGMA, composition of the triblock copolymer is DMAEMA76 -b-PEGMA48 -b-DMAEMA76 . The structure of copolymer III was shown in Fig. 1d. 2.3.3. Synthesis of diblock copolymer PDMAEMA-b-poly(PEGMA) (copolymer III) Diblock copolymer PDMAEMA-b-poly(PEGMA) was synthesized by sequential addition of different monomers into the polymerization systems. The polymerization was carried out with the ratio of [DMAEMA]/[PEGMA]/[EBiB]/[CuBr]/[HMTETA] = 100/60/1/1/1 in THF solution at 40 ◦ C. After reaction, the mixture was passed

through a basic alumina column to remove Cu complexes using acetone as the eluent. The diblock polymer PDMAEMA-b-poly(PEGMA) was obtained by removing the eluent and then precipitated in petroleum ether. 1 H NMR (400 MHz, D2 O): ı2.36 (–N(CH3 )2 ); ı2.76 (–O–CH2 –CH2 –N(CH3 )2 ); ı4.19 (–COO–CH2 –CH2 –N(CH3 )2 , –COO–CH2 –CH2 –O–); ı3.72 (–O–CH2 –CH2 –O–); ı3.36 (–O–CH3 ). GPC: Mn = 37,000, Mw/Mn = 1.20. Composition of the diblock copolymer is DMAEMA126 -b-PEGMA36 . The structure of copolymer III was shown in Fig. 1e. 2.4. Sample preparation The polymer/plasmid DNA complexes were routinely prepared by the addition of a single aliquot of the required amount of the polymer solution [20 ␮L DMAEMA solution (9.75 × 10−4 ␮g/␮L), 20 ␮L PDMAEMA solution (9.75 × 10−4 ␮g/␮L), 20 ␮L copolymer I solution (1.95 × 10−3 ␮g/␮L), 20 ␮L copolymer II solution (1.95 × 10−3 ␮g/␮L), 20 ␮L copolymer III solution (1.95 × 10−3 ␮g/␮L)] to DNA solution (10 ng/␮L, 20 ␮L), respectively to produce complexes of the desired polymer at a fixed monomer/DNA nucleotide ratio. The polymers and DNA are dissolved into TE buffer which was diluted with distilled water to the desired concentration. After incubated at room temperature for 1 h, 4 ␮L of mixed solutions were distributed on freshly cleaved mica surface. Tapping mode AFM studies were performed on a Nanoscope IIId AFM instrument (Veeco Metrology, USA) under ambient conditions and commercial silicon tips with a nominal spring constant of 40 N/m and resonant frequency of 300 kHz were used in all the experiments. 2.5. Cell culture and gene transfection HEK 293T cells (a human embryonic kidney cell line) were grown in Dulbecco’s Modified Eagles Medium (DMEM, Sigma) supplemented with penicillin/streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (FBS) at 37 ◦ C in a humidified atmosphere containing 5% CO2 and used at appropriate degrees of confluence. Cells were plated in 6-well plates at an initial density of 1 × 105 to 3 × 105 cells per well in 2 mL of working medium, the cell confluent level was controlled to make cells covered 60–70% of the plate surface before transfection. DNA–polymer complexes were formed by adding 50 ␮L of DNA (200 ␮g/mL DNA in 10 mmol/L Hepes buffer, pH 7.2) to 50 ␮L of polymer with different concentration (600 ␮g/mL, 1.8 mg/mL and 2.6 mg/mL) while vortexing. Each sample of complex contained 10 ␮g DNA and was incubated at room temperature for 20 min to form complexes. The cell-growth medium was removed and replaced with DMEM just before the complexes were added. After incubating the cells with the complexes for 4 h at 37 ◦ C in humidified 5% CO2 atmosphere, the complex-containing medium was removed and replaced with 2 mL of growth medium. After 24 h incubating, the cells were trypsinized and centrifuged at 1200 rpm. They were then washed twice with ice-cold phosphate buffered saline (PBS). Finally, they were suspended in 1 mL of PBS. The cell samples were stored on ice until

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Fig. 2. AFM images of plasmid DNA. (a) Observations show the ring structure of unfolded plasmid DNA. (b) Another ring structure of folded plasmid. The Z-scale of the AFM images is 5 nm.

further analysis was performed. Fluorescence microscopy (TE2000U, Nikon Japan) was used to monitor the expression of green fluorescent protein (GFP) in the cells. 3. Results Fig. 2 shows the AFM images of the plasmid DNA. Typical morphologies of the unfolded and folded plasmid DNA without condensing agents are revealed in Fig. 2a and b, respectively. From the aggregate topography of different condensing agents without plasmid DNA presented in Fig. 3a–e. The films with no specific characteristics and some particulates reveal that the five condensing agents could self-assemble into only featureless less-ordered structures in the experimental concentration regions. Fig. 3f–j are the typical AFM images of plasmid DNA condensates. Compared with Figs. 2 and 3a–e, it is obvious that the condensing agent/DNA complexes show different morphologies with more compact structures. It can also be observed that the structures and dimensions of a variety of condensing agent/DNA complexes are drastically different. The monomer DMAEMA could induce DNA to form global aggregates with dished pockets in the central part as shown in Fig. 4a. The high resolution image of the complex is displayed in Fig. 4b. It is obtained that the diameters of the globular structural complexes shows very broad distribution in Fig. 4c. Therefore, it is thought that

the size of DMAEMA/DNA complexes was less controllable in our experiment, even though the height indicates Gaussian distribution. The thickness of the globular aggregates is averaged to 1.6 nm (Fig. 4d). PDMAEMA is the homopolymer based on monomer DMAEMA. Other three copolymers with different structural characteristics were synthesized based on PDMAEMA. The AFM images of PDMAEMA/DNA, copolymer I/DNA, copolymer II/DNA and copolymer III/DNA complexes are shown in Figs. 5a, 6a, 7a and 8a. The appearance of the PDMAEMA/DNA complexes is similar to the one of DMAEMA/DNA complexes showing the globular aggregates from the 3D high resolution AFM image in Fig. 5b. It is observed that in Figs. 6b and 7b the morphology of copolymer I/DNA complexes is disc-like with protuberance in the center and the copolymer II/DNA complexes are some small global aggregates with dished pockets in the central part. The circular flower-like aggregates with many petals are the condensates induced by copolymer III (Fig. 8b). The diameters and heights of these complexes were determined to be 85.8 ± 6.4 nm and 2.3 ± 0.5 nm (PDMAEMA/DNA in Fig. 5c and d), 125.0 ± 34.5 nm and 2.0 ± 0.1 nm (copolymer I/DNA in Fig. 6c and d), 63.0 ± 2.7 nm and 1.5 ± 0.1 nm (copolymer II/DNA in Fig. 7c and d), 106.0 ± 13.0 nm, 0.6 ± 0.1 nm and 1.3 ± 0.1 nm (copolymer III/DNA in Fig. 8c and d). Considering the tip curvature of ca. 18 nm, the width distributions of condensing agents/DNA complexes after deconvolution are provided in Figs. 5d, 6d, 7d and 8d

Fig. 3. The aggregates of the condensing agents and the corresponding plasmid DNA condensates were observed by AFM. (a)–(e) Irregular featureless film of condensing agents observed by AFM. (f)–(j) The typical morphologies of plasmid DNA condensates. The Z-scale of the AFM images is 20 nm.

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Fig. 4. AFM images of condensates induced by monomer DMAEMA, and the statistics of the dimensions of the condensates by DMAEMA. (a) The aggregate topography of DNA induced by DMAEMA. (b) The 3D high resolution AFM image of DNA condensates. (c) The diameter distribution of DNA/DMAEMA condensates. (d) The histogram with Gaussian fitting of the height of DNA/DMAEMA condensates. The Z-scale of the AFM images is 15 nm.

Fig. 5. The morphology of condensates induced by polymer PDMAEMA, and the histograms of the dimensions of DNA condensates. (a) The AFM image of PDMAEMA/DNA complexes. (b) The 3D AFM image of DNA condensates. (c and d) The width distributions of PDMAEMA/DNA complexes without and with tip deconvolution, respectively. (e) The histogram with Gaussian fitting of the height of the complexes. The Z-scale of the AFM images is 15 nm.

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Fig. 6. AFM images of condensates induced by copolymer I, and the statistics of the dimensions of copolymer I/DNA complexes. (a) The AFM image of the DNA condensates. (b) The high resolution 3D AFM image of the DNA condensates. (c and d) The width distributions of the copolymer I/DNA complexes without and with tip deconvolution, respectively. (e) The average height of the copolymer I/DNA complexes with Gaussian fitting. The Z-scale of the AFM images is 20 nm.

Fig. 7. The condensates induced by copolymer II were observed by AFM, and the histograms of the dimensions of copolymer II/DNA complexes were revealed. (a) The morphology of the copolymer II/DNA complexes and (b) 3D AFM image of the copolymer II/DNA complexes. (c and d) The diameter distributions of the copolymer II/DNA complexes without and with tip deconvolution, respectively. (e) The histogram with Gaussian fitting of the height of the copolymer II/DNA complexes. The Z-scale of the AFM images is 10 nm.

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Fig. 8. The topography of copolymer III/DNA complexes was explored by AFM, and the statistics of the dimensions of copolymer III/DNA complexes. (a) The AFM image of the complexes. (b) The 3D AFM image of the complexes. (c and d) The dimension distributions of the copolymer III/DNA complexes without and with tip deconvolution, respectively. (e) The average height of the copolymer III/DNA complexes with Gaussian fitting. The Z-scale of the AFM images is 10 nm.

Fig. 9. The expression of GFP in HEK 293T cells illustrates the effective gene transfection using copolymer I. The mass ratios of DNA to copolymer I are 1:3 (a), 1:9 (b) and 1:13 (c).

and the diameters are 69 ± 9 nm (PDMAEMA/DNA), 102 ± 18 nm (copolymer I/DNA), 47 ± 7 nm (copolymer II/DNA) and 91 ± 12 nm (copolymer III/DNA), respectively. The double Gaussian distribution in height for copolymer III/DNA complex system is attributed to the flower-like condensate features, which may be associated with the asymmetric molecular structures. From the above AFM characterizations, it is clearly shown that the cationic homopolymer and copolymers could induce plasmid DNA to form DNA/condensing agent complexes. The gene transfection experiments were conducted in copolymer I delivery system as a typical example. The compact structures of plasmid DNA condensed by copolymer I facilitated effective gene transfection shown in Fig. 9. The green fluorescence shown in the images illustrated the successful gene transfection for expression of GFP encoded in the plasmid DNA pEGFP-C2 in mammalian cells. Effective expression of GFP for different ratios of DNA to copolymer I (Fig. 9a–c) indicated the possible adaptability of the gene delivery systems. 4. Discussion From the above AFM measurement and data analysis. It can be observed that distinct structural condensing agents could induce

DNA to form different structural and dimensional complexes. The purpose of this study was to examine the effects of different compounds, from monomer to homopolymer and block copolymers, on the dimension and morphology of DNA aggregates. The experimental results present that the dimension of aggregates can be controlled by polymers except for DMAEMA monomers in Figs. 4c, 5c, 6c, 7c and 8c. DMAEMA monomer contains a tertiary amine which is an important ingredient in the process of DNA condensation in buffer solutions. The driving force of DNA condensation induced by cationic molecules is electrostatic interaction [21–24]. DMAEMA can adequately neutralize the electronic repulsion of the DNA phosphate backbone and then induce DNA condensation. However, it is not controllable of the nitrogen amount which reacted with the plasmid DNA so that the dimension of DMAEMA/DNA complexes is also less controllable. In contrast, polymers contain quantitative nitrogen atoms and there is a quantitative restriction of nitrogen in polymers when it interacts with plasmid DNA so that the dimension of polymer/DNA complexes could be statistically optimized, showing Gaussian distribution. Thus, more uniform and regular aggregates could be formed using polymer condensing agents rather than DMAEMA monomers.

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It is also demonstrated from our results that the dimension of the aggregates is affected by chemical composition of polymers. In our experiment, the fixed gross amount of nitrogen in polymers and fixed mass of DNA are necessary in that the dimensions of condensates are strongly affected by the different N/P ratios. The other two factors in DNA condensation are the molecular weight (MW) and cationic distribution of different condensing agents. Therefore, based on the two factors, we introduced the concept of nitrogen mass per polymer unit. (The definition of nitrogen mass per polymer unit in our experiment is the mass of the nitrogen divided by the polymer MW.) As Wolfert et al. reported [33], molecular weight of polymers is keen to the size of the complexes. The polymers with higher MW would result in the larger dimensional complexes. In this work, the MW of the polymers we used is shown below (PDMAEMA: 15,700; copolymer I: 15,500; copolymer II: 47,000; copolymer III: 37,000). In the three copolymers, the PDMAEMA portion accounts for 50% of the total molecular weight. Poly-(PEGMA) accounts for the other 50% of the total MW. The poly-(PEGMA) fragment not only prevents the aggregation due to the steric hindrance, but also decreases the nitrogen mass per polymer unit. Thus, it is concluded that the ratio of nitrogen mass per polymer unit is shown as following: copolymer I:copolymer III:PDMAEMA:copolymer II = 1:1.67:2:3. The ratio of the dimension of aggregates adjusted by considering tip convolution effect induced by condensing agents is presented below: copolymer I/DNA complexes:copolymer III/DNA complexes:PDMAEMA/DNA complexes:copolymer II/DNA complexes = 102:91:69:47. It is suggested from the two ratios above that the higher nitrogen mass per polymer unit could result in smaller DNA aggregates. In view of the polymer MW, the effect of nitrogen mass per polymer unit is a more precise factor in DNA condensation, especially for the systems with copolymers. Furthermore, it is very important to investigate the effect of poly-(PEGMA) portion in polymer condensing agents. It might affect the dimension of the complexes, and even modulate the structure of the DNA aggregates. Poly-(PEGMA) contains hydrophilic polymer side chains poly (ethylene glycol). It may increase the biocompatibility of the condensing agents, while decrease the interaction of complexes with other ionic molecules. The AFM results above demonstrate that the morphology of the polymer/DNA complexes is distinct owing to the poly-(PEGMA) effect. The different structures resulting from positions of poly(PEGMA) in copolymers might regulate the topography of DNA condensates. Furthermore, the interaction between the poly(PEGMA) and plasmid DNA need to be further explored. It is inferred that the multiple intermolecular interaction is involved in the process of DNA condensation, such as electrostatic interaction, hydrogen bonding interaction and van der Waals interaction. Therefore, we should get insight into the relationship between the structure of DNA aggregates and copolymers, as well as the diversity of interactions in the process of condensation. Further understanding of the interactions in DNA condensation will aid the development and optimization of gene delivery vectors. The dimensions of condensates obtained from AFM measurements in air are mostly distinct from the ones from DLS method in liquid as revealed in a number of previous reports [8,17,34]. The difference could be largely attributed to the environments where AFM and DLS measurements are performed, as well as the detection mechanisms. The results from DLS method reveal three-dimensional characteristics of the condensates suspended in solution and AFM provides the morphology features of the condensates on surfaces. Common to these methods is the interactions between DNA and condensing agents. It is therefore plausible that trend of condensate dimension induced by different agents observed from AFM is complementary to the one obtained from DLS [8]. It is also worth noticing that the measured width of the

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aggregates maybe interfered by the convolution effect of AFM tip geometry and could be qualitatively adjusted [34]. The convolution effect could be examined by equation W2 = 8RH to calculate the diameter of objects observed by AFM considering the tip convolution effect. (W: the width of objects observed by AFM, R: the radius of tip, H: the height of the objects shown in Fig. S1a) The width of plasmid DNA is measured to be 17 nm (W = 17 nm), and expected diameter of DNA is 2 nm (H = 2 nm), thus, the radius of tip is approximately 18 nm (R = 18 nm) based on the equation W2 = 8RH. The artificial width (Wart ) is obtained by equation R2 = (R − H)2 + (Wart /2)2 shown in Fig. S1b. The width distributions adjusted by considering the tip convolution effect are provided in Figs. 5d–8d. In the gene transfection experiment, we initially select copolymer I to transfect genes and the results show that the DNA/copolymer I complexes could go into cell by endocytosis. The dimension of DNA/copolymer I complexes is larger than others and it might be easier to occur endocytosis than other smaller DNA condensates. The gene transfection experiments of other agents will be undertaken further. 5. Conclusion By using atomic force microscopy, the different DNA condensation structures were studied for distinct polymeric condensing agents. It could be concluded that (1) the size of the aggregates could be modulated by polymers rather than the monomer, in the process of DNA aggregation; (2) in case of cationic polymers as condensing agents, dimension of DNA condensates is sensitive to the nitrogen weight per polymer unit comparing with the MW of polymers, which is the one with higher nitrogen mass per polymer unit could induce DNA to form smaller condensates; (3) poly-(PEGMA) has the effect on regulating the structure of DNA aggregates, especially for copolymer I, copolymer II and copolymer III. We believe that the AFM results in this work reveal the self-assembling mechanism of cationic polymer/DNA aggregates to a certain extent, and the gene transfection results could also be beneficial to the development of gene delivery and therapy in the future. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant number: 90403140) and the National Basic Research Program of China (2009CB930100). Financial support from CAS Key Laboratory of Nano Bioeffect and Biosafety is also gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfb.2009.08.040. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]

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