Filamentous Condensation of DNA Induced by Pegylated Poly-l-lysine

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Biomacromolecules 2008, 9, 2048–2055

Filamentous Condensation of DNA Induced by Pegylated Poly-L-lysine and Transfection Efficiency Vesna Stanic´,*,† Youri Arntz,† Doriane Richard,† Christine Affolter,† Isabelle Nguyen,† Corinne Crucifix,‡ Patrick Schultz,‡ Corinne Baehr,§ Benoit Frisch,§ and Joelle Ogier† INSERM UMR 595, Institut National de la Sante´ et de la Recherche Me´dicale, UFR d’Odontologie, Universite´ Louis Pasteur, F-67085 Strasbourg, France, IGBMC, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, 1 rue Laurent Fries, F-67404 ILLKIRCH Cedex, France, and IGL, Institut Gilbert Laustriat, UMR 7175 Laboratoire de Chimie Enzymatique et Vectorisation, Faculte´ de Pharmacie, F-67400 ILLKIRCH Cedex, France Received March 19, 2008; Revised Manuscript Received May 10, 2008

In this paper we propose a detailed analysis of structural and morphological properties of two poly-L-lysine (PLL)based transfection formulations, PLL/DNA and pegylated PLL (PLL-g-PEG)/DNA, by means of atomic force microscopy (AFM) and transmission electron microscopy (TEM). Comparing PLL-g-PEG/DNA with PLL/DNA polyplexes, we demonstrate that, due to the presence of PEG, the particles differ not only in size, shape, and crystalline structure, but also in transfection effciency. While PLL condensates DNA in large agglomerates, PLL grafted with polyethylene glycol 2000 can condensate DNA in long filaments with diameters of some nanometers (6-20 nm). These structures are dependent on the grafting ratio and are more effcient than compacted ones, showing that DNA uptake and processing by cell is directly related to physicochemical properties of the polyplexes.

1. Introduction Experimentation of various synthetic polymer DNA carriers in gene therapy still attracts a big interest because of their potentiality to substitute viral transfection.1–12 However, low transfection efficacy and cytotoxicity still remain a problem in these approaches. To improve transfection formulations, understanding the structural properties in relationship with the surface functionality and the interfacial properties of the complexes is crucial and would help to delineate structural advantages for an optimized transfection efficiency. It has been previously demonstrated that DNA can be complexed with poly cationic substances such as poly-L-lysine (PLL), poly-L-ornithin, or polyethylenimine.13–17 Although polymers with high cationic density condense plasmid DNA into structures amenable to cellular internalization via endocytosis, the high charge density is one factor that contributes to their cytotoxicity.18,19 On the other hand, low cationic charge density can reduce or eliminate DNA condensation capability. The balance between cationic charge density and DNA condensation is complicated even further when endosomal escape moieties and nuclear translocation sites are considered. To circumvent the inherent cytotoxicity of, for example, PLL-forming DNA nanoparticles, grafted copolymers of PLL and polyethylene glycol (PEG) were designed.20 Moreover, pegylation should improve the solubility and stability of PLL-g-PEG/DNA complexes in aqueous solution and steric effects of exposed PEG chains should result in nanoparticles with reduced nonspecific protein adsorption as compared to charged polymer-DNA complexes.21 In addition, pegylation might contribute to their cellular uptake.19,21 It is well-known that particle size is a key parameter in the process of cellular uptake and it has been shown * To whom correspondence should be addressed. E-mail: vesna.stanic@ medecine.u-strasbg.fr. † INSERM UMR 595. ‡ IGBMC. § IGL.

that nanometric carriers are able to overcome biological barriers and even penetrate well-organized mucosae.1 Moreover, submicrometric particles can be administered intravenously, which could provide additional advantages for applications such as anticancer therapy. The structure of the copolymers in solution is strongly dependent on their molecular weight and their relative composition.22 Furthermore, the role of the molecular weight (MW) of the PEG chains in the formation of polyplexes has been recently enlightened in PEG-PEI-antisense oligoribonucleotide systems.22 The combined physicochemical data, reported by Glodde et al. 2006,23 provide the sufficient information to construct the theoretical models of the polyplex structures, although the exact details remain uncertain. In this paper we report detailed results of atomic force microscopy and transmission electron microscopy. The aim is to clarify the relationship between polymer/DNA complexes structure and cell transfection effciency. We show how structural and morphological properties of complexes influence transfection effciency. We compare PLL/DNA and PLL-g-PEG/DNA polyplexes, demonstrating that due to the presence of the PEG chain in polyplexes, DNA can condensate in long “spaghetti-like” filament, which thickness is dependent on the grafting ratio. The filament shape, rather than PLL/DNA macro particles, seems specifically to be preferable for transfection of Cos1-cells.

2. Materials and Methods 2.1. General Procedure for the Synthesis of PLL-g-PEG Copolymers. High molecular weight (MW) poly-L-lysine hydrobromide (PLL, MW ) 15-30 KDa, 71-142 repeating units) and 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) were obtained from Sigma Aldrich (Saint Quentin Fallavier, France). Methoxy-PEG acid (PEG, MW ≈ 2000 g/mol, 41 repeating units) was obtained from Iris Biotech (Marktredwitz, Germany). N-Hydroxysulfosuccinimide (sulfoNHS) was from Fluka (Saint Quentin Fallavier, France). Boric acid was from

10.1021/bm800287z CCC: $40.75  2008 American Chemical Society Published on Web 06/24/2008

Filamentous Condensation of DNA

Figure 1. Schematic representation of synthesis of PLL-g-PEG copolymer.

Merck (Fontenay sous bois, France). Macroprep High S support, a strong cationic exchange support was from Biorad (Marne-la-Coquette, France). Schematic representation of the PLL-g-PEG copolymers is shown in Figure 1. Different types of PLL-mPEG with varying PEG contents were synthesized. Synthesis of 9 mol % PLL-mPEG is described as an example. The mPEG (191 mg, 0.095 mmol) was activated with 1-ethyl3-(3-dimethylaminopropyl)carbodiimide (21 mg, 0.114 mmol) in a borate buffer (0.5 M, pH 8.4, 15 mL). After stirring 1 h at room temperature, N-hydroxysulfosuccinimide (25 mg, 0.114 mmol) and poly-L-lysine (80 mg, 0.38 mmol) were added. After stirring for 3 days at room temperature, the mixture was neutralized to pH 4-5 with aqueous HCl 1 N. The reaction medium was then purified through a strong cation exchange column (sodium salt, 160 ( 40 µequiv/mL). The first elution with deionized water gave free PEG, a second elution worked with aqueous NaCl 1 M and then with 5 M NaCl to isolate the grafted PLL-g-PEG. After elimination of byproduct by dialysis against 2 × 5 L of deionized water, the solvent was eliminated by lyophilization and gave 74 mg of PLL-g-PEG copolymer with 9% PEG. The NMR characterization was performed with a 300 MHz Bru¨cker spectrometer (DPX 300). 1 H NMR (300 MHz, D2O, reference tert-butanol 1.23): 1.43 1.69 (m, -CH2CH2CH2CH2NH-, 633H), 2.5 (s, -NHCOCH2CH2CONH-, 36H), 2.99 (m, CH2CH2CH2CH2NH-, 223H), 3.37 (m, -OCH3, 50H), 3.69 (s, -CH2OCH2-, 1006H), 4.29 (s, CH-, 100H). The other PLL-g-PEG copolymers were obtained using the same procedure but changing the ratio between PLL and PEG molecules. For instance, 0.1 equiv PEG gave 4.5% grafting, 0.15 equiv PEG gave 6% grafting, 0.3 equiv PEG gave 19% grafting, and 0.6 equiv PEG gave 40% grafting. The calculated net positive charges of PLL and pegylated copolymers used in our experiment are as follows: =106 for PLL, and =102, =99, =96, and =63 for PLL-mPEG at grafting ratios 4.5, 6, 9, and 40%, respectively. 2.2. Preparation of the Complexes. Plasmid DNA pEGFPC1(Clontech BD Science) expressing enhanced green fluorescent

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protein, with total size of 4700 base pairs ≈ 1.5 µm contour length, was used for all microscopy experiments. For cell transfection experiments, plasmid DNA pIV1066, expressing firely luciferase (gift from Dr. C. Schuster), with a comparable size as pEGFP-C1, was used. The plasmid DNA was amplified in XL1 blue strain of E. coli and extracted by the standard process. Linear pEGFP-C1 DNA used for microscopy experiments was obtained by enzymatic treatment with BamHI. After linearization, the plasmid DNA has been precipitated with ethanol and analyzed by agarose gel electrophoresis with Tris borate (TBE) 1%. The PLL-g-PEG copolymers with various grafting ratio used for microscopy experiments are shown in Table 1. All samples were prepared in buffer saline solution (4-(2-hydroxyl) piperazine-1-ethanesulphonic acid (HEPES), 20 mM, pH ) 7.4 and NaCl 150 mM). For cell transfection experiments, 1 µg DNA dissolved in 100 µL saline Hepes buffer was added into the polymer solution. Assuming that 1 µg DNA contains 3 nmol of anionic phosphate, copolymers were diluted to the appropriate concentration of residual free nitrogen atom to produce given N/P ratios. 2.3. Atomic Force Microscopy (AFM). The images were performed with a commercial AFM microscope Nanoscope IV from Veeco (Santa Barbara, CA). The experiments were made in tapping mode both in air and liquid. The samples in air were prepared at various weight ratios. A drop of the sample (15 µL) was deposited onto SiO2 wafer followed by a 5 min adsorption step. Excess of fluid was removed with a filter paper, and the samples were then dried under a vacuum dryer for 12 h. The samples in liquid were deposited on fresh cleaved mica surface and observed with a Nanoscope specific liquid cell. The cantilevers (Nanoworld company, Neuchatel Switzerland) used in air measurement and, respectively, in liquid measurement were FMR, with a resonance frequency of 75 kHz and a spring constant of 2.8 N/m and, respectively, NCHR, with a resonance frequency of 300 kHz and a spring constant of 42 N/m. Images were acquired with a resolution of 512 × 512 pixel with a scan rate of 3 Hz. 2.4. Transmission Electron Microscopy (TEM). For negative stained samples, all the PLL-g-PEG/DNA complexes were diluted by a factor of 10 and, in the case of the DNA samples, by a factor of 20. Drops (5 µL) of each preparation were placed on a 10 nm thick carbon film previously treated by glow discharge in amylamine. After a 2 min adsorption, the grid was negatively stained with a 2% uranyl acetate solution. In the case of DNA samples, we performed an additional evaporation of platinum to enhance the contrast and it is done with the rotational shadowing technique. Micrographs of negative stained samples were recorded on Kodak SO163 photographic plates at a magnification of 28000× using transmission electron microscope operating at 100 kV with a LaB6 filament (model CM120, FEI). To perform the measurements, the micrographs were digitized on a drum microdensitometer (primescan D7100, HEIDELBERG) at 5 µm raster size, resulting in a pixel spacing of 0.18 nm. For Cryo-TEM, the samples were used nondiluted. A total of 5 µL of the preparation was placed on a holey carbon foil grid and then blotted for 5-10 s with a filter paper and plunged into an ethane slush cooled with liquid nitrogen. The cryo-samples were imaged in a cryoelectron microscope operating at 200 kV and equipped with a field emission gun (model Tecnai F20G2, FEI). Micrographs were recorded on Kodak SO163 photographic plates at a magnification of 28 000 X using transmission electron microscope operating at 100 kV with a LaB6 filament (model CM120, FEI). To perform the measurements, the micrographs were digitized on a drum microdensitometer (primescan D7100, HEIDELBERG) at 5 µm raster size resulting in a pixel spacing of 0.18 nm. 2.5. In Vitro Transfection. 2.5.1. Cell Culture. Cos-1 cells (ATCC number CRL 1650) were maintained in DMEM medium supplemented with 10% FCS (fetal calf serum) and 0.5% antibiotics in a 5% CO2 incubator. For the transfection study, cells were seeded at a density of 105 cells per well in a 24-well plate and incubated 24 h before the addition of the plasmid DNA/polymer complex.

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Table 1. Characteristics of Synthesized Copolymers Used for Comparisona

a

copolymer

PLL MW [KDa]

PEG MW [KDa]

copolymer MW [KDa]

g [%]

DNA [Kbp]

PLL-g-PEG PLL-g-PEG PLL-g-PEG

15-30 15-30 15-30

2 2 2

21.0-42.7 27.7-55.5 71.8-143.6

4.5 9 40

4.7 (circular/linear) 4.7 (circular) 4.7 (circular)

MW values of copolymers are based on MW of PLL and PEG provided by supplier. g ) PEG grafting ratio.

2.5.2. Transfection Protocol. Plasmid pIV1066/PLL or plasmid pIV1066/PLL-g-PEG complexes were prepared by mixing 1 µg of plasmid and various amounts of polymer in 100 µL of HEPES (20 mM)/NaCl (0.15 M) buffer and incubated for 15 min at room temperature. Medium from each well was removed and replaced by 500 µL of DMEM medium without serum. After 15 min, 100 µL of transfection mixture was added on cells. Cells were then incubated for 4 h at 37 °C in a 5% CO2 incubator. After 4 h, the transfection mixtures were removed and 1 mL of fresh medium with serum was added to each well. Cells were incubated for an additional 48 h before measuring the luciferase activity in each well. 2.5.3. Luciferase Assay. The effect of different N/P ratio on transfection effciency was analyzed with the luciferase assay system (Promega). Briefly, cells were lysed with PBS/Triton X100 0.1%. Cell lysate (20 µL) was mixed with 100 µL of luciferase assay system and luminescence was measured on a luminometer. The results were normalized by measuring quantity of protein per sample to express results as RLU (relative light unit)/mg of protein. 2.5.4. Viability Assays. The measurement of viability is done by the acid phosphatase method which consists in the titration of the enzyme whose activity varies with the number of viable cells. Cells were treated in the same way as for transfection assay. After 48 h of culture, supernatant was discarded and cells washed with PBS. Then 600 µL of substrate consisting of 10 mmol/L of p-nitrophenylphosphate (pNPP; Sigma) diluted in sodium acetate buffer was added to each well. The plates were incubated at 37 °C during 1 h under humidified atmosphere. The reaction was stopped by adding 10 µL of 1 N sodium hydroxide. The absorbance of the yellow colored solution was measured at 405 nm using a plate reader. The percentage of viability was established using Cos-1 in normal culture conditions as control (i.e., absorbance of control corresponds to 100% of viability).

3. Results 3.1. PLL-mPEG Copolymers Synthesis. PLL-mPEG copolymers synthesis was achieved by coupling linear mPEG blocks to a linear PLL macromolecule through an amide bridge. The single acidic terminal group of mPEG was activated in situ with EDC and N-hydroxysulfosuccinimide, prior to the coupling reaction with the PLL macromolecule in a borate buffer (see Figure 1). After the reaction, the polymers were purified through a strong cation exchange column and then dialysed few times against water to remove impurities. The purity and structural authenticity were confirmed with 1H NMR. The number of mPEG chains grafted onto a PLL backbone was calculated from the relative integration ratio of 1H NMR peak from the proton of the asymmetric carbon on the lysine at 4.29 ppm (1H) and the peak at 2.5 ppm (4H) from the PEG unit. From this method, the PEG content ratios of the polymers were determined as 4.5, 6, 9, 19, and 40 mol %, respectively. 3.2. Atomic Force Microscopy (AFM) Analysis. AFM can be an excellent technique to obtain details at structural and even molecular level. In our case, by applying the Tapping Mode where the tip of the AFM touches the sample only slightly, it was possible to visualize the samples with high resolution. We report results that elucidate the structural and morphological differences of PLL/DNA and PLL-g-PEG/DNA complexes, in air as well as in solution. Only PLL-g-PEG/DNA (g ) 4.5%

and g ) 40%) were selected for AFM study to compare two extremes. Figure 2A shows AFM images in air of PLL-g-PEG copolymer, g ) 40% on silicon wafer substrate in air. Copolymer seems to be collapsed in particles of different shape and size (range of particle size are from some nm to some hundred nm). Similar morphology was observed for the PLLg-PEG copolymer with low grafting ratio g ) 4.5%. Guo et al. (2004)24 provided AFM images in air for nanoscopic particles of PEG7000-g-PLL/DNA and PEG20000-g-PLL/DNA polyplexes showing networks of molecular structures on the surface. According to the preparation procedure described in Materials and Methods, we observe a similar network organization for the PLL-g-PEG/DNA complexes (Figure 2B). Comparison of the PLL-g-PEG and PLL-g-PEG/DNA micrographs shows that this network organization is induced by the complexation of DNA with PLL-g-PEG. Networks of bundles connected with long chains suggest that DNA condensates in filamentous polyplexes. Now if we compare PLL-g-PEG/DNA complexes with PLL/ DNA complexes (Figure 2B,C), it is visible that PLL/DNA complexation gives more particle aggregates and is bigger in size. In the corner of each figure is represented a magnification of one region, putting in evidence better details and morphology of the surface. Magnification of Figure 2C suggests some columnar phase ordering for the PLL/DNA complexes. A crystalline ordering system is even indicated by a geometrical form of particles. Because the water is totally evaporated from the polyplexes, all structures are collapsed and attached on the surface. AFM imaging in aqueous solution was necessary to avoid those problems. In Figure 3 are shown AFM micrographs in liquid. Figure 3A represents the surface of PLL/DNA complexes, while Figure 3B,C represents PLL-g-PEG/DNA at grafting ratios of g ) 4.5% and g ) 40%, respectively. Long filamentous structures with diameters ≈15-20 nm were detected for the grafting ratio g ) 4.5% and a very thin filament ≈6 nm for the grafting ratio g ) 40%. This kind of filament, however, is not observed for the PLL/DNA complexes in the absence of PEG (Figure 3A). Unlike the dried state, filaments in aqueous solution are separate, and fluctuated objects and network organization is less evident and less rigid. DeRouchey et al. (2005)25 and Castelletto V. et al. (2006)26 already described PLL/DNA polyplexes in water as white fluctuated aggregates. PLL/DNA aggregates look here like transparent films, approximately 20 nm in thickness. When the two complexes in HEPES buffer at the same salt concentration are compared, it is evident that polyplexes with PEG have a filamentous shape and that there are no macroagglomerates of particles, as observed in the absence of PEG. PEG chains act to screen electrostatical forces in the formation of PLL-g-PEG/DNA complexes and probably circumvent interactions between the complexes making the all system more homogeneous and balanced. The PLL-g-PEG/DNA complex solution is transparent, indicating isotropically distribution of fluctuated filaments. It seems that PEG controls the size of the complexes and has a large influence in the aggregation process. The grafting ratio of PLL-g-PEG copolymers plays also an


Figure 2. AFM micrographs in air. (A) PLL-g-PEG adsorbed on SiO2 surface at grafting ratio g ) 40% (N/P ) 12.7), with magnification of spots in one region. (B). PLL-g-PEG/DNA complexes adsorbed on SiO2 surface at the same grafting ratio showing the typical network organization of complexes. In the corner is magnification of one network spot from the image. (C) PLL/DNA complexes adsorbed on SiO2 surface: image represents the surface of big condensate of PLL/ DNA complexes; magnification of details showing the columnar phase.

important role for the size and shape of polyplexes,22,23 but it has to be mentioned that preparation of samples is one of the


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Figure 3. AFM micrographs in liquid. (A) PLL/DNA complexes adsorbed on mica: surface of big PLL/DNA complexes condensate in water; (B) PLL-g-PEG/DNA complexes adsorbed on mica at grafting ratio g ) 4.5% (N/P ) 8) showing evidence of separate filaments with diameter approximately 20 nm; (C) PLL-g-PEG/DNA complexes adsorbed on mica at grafting ratio g ) 40% (N/P ) 12.7) showing very thin filaments, approximately 6 nm.

limit of AFM techniques and we did not observe with this technique any difference between the complexes with the high and the low grafting ratio. 3.3. Transmission Electron Microscopy (TEM) Analysis. The stiffness of a polymer and the attractive forces between its chain segments relative to the interaction between chain

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Figure 4. Negative stained TEM micrographs. (A) TEM micrographs of super coiled DNA for comparison; (B) PLL-g-PEG/DNA complexes at grafting ratio g ) 4.5% (N/P ) 8) showing one example of filament overlapping with diameter of filament around 20 nm; (C) PLL-g-PEG/DNA complexes at grafting ratio g ) 9% (N/P ) 8.45) showing filament overlapping with evidence of thin chains around big filaments with diameter of filaments around 8 nm; and (D) PLL-g-PEG/DNA complexes at grafting ratio g ) 40% (N/P ) 12.7), filaments overlapping with a diameter of approximately 4-6 nm.

segments and the solvent could predict transition of a polymer from a random coiled to a compact structure.27,28 The existing theoretical models of this process are not specific for DNA. In fact, Tang M.X. et al. (2005)29 showed toroid formations for non-DNA polymers. Detailed evaluation for PLL/DNA complexes was performed by Liu G. et al. (2001).30 They describe the effect of the salt concentration on the mode of binding of PLL and DNA demonstrating that, at low salt concentration, the complexes can take toroid form. We examined the influence of grafting ratio on the PLL-g-PEG/DNA structure formed at fixed salt concentration by means of negative-stained TEM technique. We systematically explored the influence of the PEG chain in the formation of filament structures. For comparing with PLL-g-PEG/DNA complexes, we have included one image of supercoiled DNA conformation, Figure 4A. The structure of PLL-g-PEG/DNA polyplexes at grafting ratio, g ) 4.5%, g ) 9%, and g ) 40%, is shown in Figure 4B-D. As already mentioned before with AFM analysis, pegylated PLL induces filamentous disordered isotropic structures when complexed with DNA rather than compact aggregates with crystalline hexagonal ordering in the case of PLL/DNA complexes, as suggested by DeRouchey J. et al. (2005).25 Experiments that we carried out with small angle X-ray scattering, on the PLL-g-PEG/DNA complexes, confirmed also that there is no crystalline ordering (manuscript in preparation23). Long filament structure of the complexes indicates that DNA molecules are stretched and coated with PLL-g-PEG copolymer. In Figure 4B are reported PLL-g-PEG/DNA complexes with linear DNA at g ) 4.5%, where is clearly visible the turn of the filaments with loops on the extremes. Several filaments are turned together as a “spaghetti-like” structure following the spiral shape of DNA. The negative stained-TEM image in Figure 4C clearly indicates morphology around PLL-g-PEG/DNA complexes showing details of thin chains (diameter around 2 nm), which could be attributed to free PLL-g-PEG chains.

According to the results of complexes electrophoretic mobility onto agarose gels, there is no free DNA molecule in solution. The maximal diameter of thick filaments varies from 6 to 20 nm according to grafting ratio. If we consider that diameter of dsDNA is 2 nm, it means that 3-10 molecules may overlap together according to obtained diameter range (6-20 nm). In Figure 4C is shown an example of filaments with a diameter of about 9 nm at grafting ratio g ) 9%, the sample having been prepared in HEPES and NaI salt buffer to obtain better contrast of small chains surrounding the big filaments. With higher grafting ratio, g > 9%, filaments are thinner, approximately up to 6 nm (Figure 4D), which would correspond to overlapping of 2 or 3 molecules. Considering that overlapping of filaments follows the shape of DNA, we can give one approximation of size of the filaments pitch. One turn of dsDNA corresponds to 10.5 base pairs that correspond to 3.57 nm. The pitch of one turn in filaments varies from 20 to 40 nm as a function of the number of overlapped DNA molecules. Figure 5B displays in 3D view the image represented in Figure 5A, putting in evidence better details of filaments and background of image. With 3D view image, we discriminate better organization of filaments, showing how from thinner they overlap in thicker filaments. The peaks along the filaments represent the filaments pitch (white color corresponds to maximum of intensity). Linear and circular DNA was compared, but there is no evident difference in form and size between the structures of the two complexes (Figure 4B and Figure 4D). Direct proof from imaging of the filaments was confirmed also by cryo-TEM experiments, (Figure 6) with the size and shape of filaments confirming exactly results reported previously by AFM and negative staining TEM experiments. Table 2 summarizes the results obtained with the different microscopy approaches. There is given maximal diameter size of filaments as a function of grafting ratio.



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Figure 5. 3D view of PLL-g-PEG/DNA filaments at grafting ratio g ) 40% (N/P ) 12.7), giving the better vision of small chains around the filaments.

Figure 6. Cryo-TEM micrographs. (A) PLL-g-PEG/DNA complexes at grafting ratio g ) 4.5% (N/P ) 8) showing filaments overlapping with diameter of 20 nm; (B) PLL-g-PEG/DNA complexes at grafting ratio g ) 9% (N/P ) 8.45) showing filaments with diameter of filaments around 8 nm; (C) PLL-g-PEG/DNA complexes at grafting ratio g ) 40% (N/P ) 12.7) showing filaments overlapping with diameter of approximately 6 nm. Table 2. Summarized Diameter Size for the Filaments Obtained by Means of AFM and TEM Microscopy Measurements PLL-DNA

agglomerates g 1 micron

PLL-PEG-DNA, g ) 4.5 PLL-PEG-DNA, g ) 9 PLL-PEG-DNA, g ) 40

=20nm =9 nm =6 nm

3.4. In Vitro Transfection. In vitro transfection effciency of Cos-1 cells was evaluated using the pIV1066 plasmid DNA. Figure 7 shows the transfection efficiency of different N/P formulations (3, 6, 12, 15, 18, 21, 28, 36, 44) of PLL-g-PEG/ pIV1066 complexes in Cos-1 cell lines for g ) 4.5%. It is confirmed here that plasmid DNA complexed with nonpegylated PLL has a very low transfection efficiency.31,32 The transfection effciency of the PLL-g-PEG/DNA group is at least five times higher than the transfection effciency of the PLL/DNA group. These results have to be put in relation with the cell viability curves comparing cytotoxicity of pegylated and nonpegylated complexes (Figure 8). Viability of the Cos-1 cells was tested to explore possible cytotoxicity of the complexes formed at various N/P ratios. When comparing the two types of complex namely PLL/DNA and PLL-g-PEG/DNA at g ) 4.5%, the biocompatibility of PLL/ DNA complexes for Cos1-cell for N/P above 5 drastically decreases and becomes less than 10% which makes null the transfection rates in this N/P value domain. In contrast, the cell viability for the PLL-g-PEG/DNA carrier is high even for very high N/P ratio, in the range of 70% to 90% depending on N/P.

Figure 7. Comparison of transfection of PLL-g-PEG/DNA and PLL/ DNA complexes in Cos-1 cells with various N/P ratios. (b) represents the transfection of PLL-g-PEG/DNA at grafting ratio g ) 4.5%; (O) represents the transfection of PLL/DNA complexes. Two independent experiments have been performed.

Those results confirm that PEG increases polymer biocompatibility. The effect of the grafting ratio on transfection effciency is depicted in Figure 9 for different N/P ratios. The highest transfection effciency was observed for g < 9%. Considering the infiuence of the grafting ratio on DNA complexation with PLL-g-PEG, namely on the size parameters of the filaments, grafting ratio of 9% being the threshold between thicker and thiner filaments, this result indicates that increased overlapping of DNA molecules within filaments (it means

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Figure 8. Cytotoxicity for pegylated versus nonpegylated complexes in the Cos-1 cells: (•) 4.5% grafted PLL-g-PEG/DNA complexes and (O) PLL/DNA complexes. The continuous lines are guides for the eyes. Two independent experiments have been performed.

Figure 9. Comparison of transfection of PLL-g-PEG/DNA complexes in Cos-1 cells at various grafting ratio. Three independent experiments have been performed and statistics performed by using the Student test.

increasing condensation of DNA) resulting in these typical diameter size up to 20 nm is in favor of cell transfection and that at least filament structures are much more effcient in transfecting Cos-1 cells than big aggregates.

4. Discussion Understanding how structure of gene delivery particles may influence transfection efficiency is a key step in optimization of particle design. In this work, we investigate structural and morphological properties of two PLL-based polyplexes and their correlation to transfection efficiency. Comparing PLL/DNA and PLL-g-PEG/DNA polyplex, we demonstrate that, due to the influence of PEG, these polyplexes are different not only in size, shape, and crystalline structure, but also in total transfection efficiency. That pegylation of gene delivery particles significantly affects transfection efficiency and particularly cellular uptake and intracellular trafficking of particles that had previously been reported (Mishra et al. 2004),33 but the novelty here is that we further show that it may be related, for PLL-based polyplexes, to specific filamentous DNA condensation generated by a given PEG grafting ratio range for given PEG and PLL chains lengths. We show that pegylation of linear PLL hampers the condensation in big particles and their agglomeration and that the presence of PEG in polyplex induces the formation of filament forms, which are specifically efficient for transfection. Our

Stanic´ et al.

experimental results show, namely, for the first time to our knowledge, that pegylation determines open structures forming turned filaments following the DNA spiral conformation. With increasing of the parameter g, diameter of filaments decreases, probably because of the higher quantity of PEG grafted to the PLL chain that may restrain DNA molecules inside and PEG chain outside of filaments, circumventing the filaments overlapping. This may be due also, at least in part, to the concentration of DNA related to N/P ratio. With higher grafting ratio in complexes, the filaments are probably totally coated with PEG and more separated from each other. Length and flexibility of the grafted PEG moieties probably control stealth properties of the condensates. Studies that used PEG of 5 or 12 kDa report that cell viability and transfection efficiency were similar with 5 or 12 kDa PEG at 5 or 10% grafting,20 but that high PEG grafting (g20%) transfects poorly even with PEG 5 kDa. It suggests that PEG with higher MW, like high PEG grafting ratio, would hamper stability of condensates. Mao et al. (2006)33 observed aggregation of PEIg-PEG/siRNA complexes in case of lower molecular weight of PEG (MW 550). They observed that the phenomenon of aggregation decreased with increasing PEG chain length to 2 and 5k. These results are in agreement with our hypotheses also. With higher grafting ratio, interaction between polymer and DNA is reduced because the positive charges of PLL are screened. At the same time, PEG located on the external part of filaments protects complexes from outside. We demonstrate that the difference induced by pegylation at some level in physicochemical properties of poly-L-lysine may have a great impact on transfection effciency, thus probably on cellular uptake and polyplex stability in cell compartments. We demonstrate that filaments diameter size of 20 nm obtained at lower grafting ratio (g < 9%) is in favor of cell transfection, probably as a result of the balance between cationic charge density of PLL-g-PEG and DNA condensation. The filamentous structure might produce a balanced interaction between copolymer and DNA and might play a positive role in DNA decondensation to allow gene expression. Besides, these results are in accordance with recently obtained results by M. Walsh et al. (2006),34 on C1K30-PEG complexes, reporting that the diameter of rod-like pegylated poly-L-lysine/DNA nanoparticles is likely to influence the mode of cellular entry of the complexes. They show that the pegylated polymer is capable of compacting plasmid DNA into toroid-like and rod-like nanoparticles having an average width of 20 nm, potentially allowing the nanoparticles to cross the nuclear membrane pore and to transfect nondividing cells. They demonstrate that C1K30-PEG vectors have significantly higher transfection efficiency than unpegylated C1K30. This experiment totally confirms our results obtained for transfection effciency with pegylated and unpegylated PLL polyplexes. Our conclusion is that PLL-g-PEG/DNA complexes due to filament shape of the particles and their good biocompatibility produce higher effciency of transgene expression than PLL/ DNA particles. Specially, filament dimensions of PLL-g-PEG/ DNA complexes seem to be favorable for cell uptake, complex processing, and finally, transfection efficiency. In this regard, our results could support future rational design of vectors to improve the efficiency of gene delivery. Acknowledgment. It is a pleasure to thank Dr. Heike Hall and Pr. Marcus Textor (Dept. Materials, ETH Zurich) for the critical reading of article and preliminary discussion, respectively. We acknowledge the “Conseil Scientifique de l’Universite´ Louis Pasteur”, the Dental Faculty of Strasbourg and the


Canceropole Grand Est-Fond National pur la Sante´ ACI 2004 for financial support.

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BM800287Z

Filamentous Condensation of DNA Induced by Pegylated Poly-l-lysine

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