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Oct 22, 2008 - Such star polymers, which allow the binding and release of DNA, ..... All star polymers P2, P4, and P6 bear free primary amino groups, ...
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Biomacromolecules 2008, 9, 3231–3238

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Dendritic Star Polymers for Efficient DNA Binding and Stimulus-Dependent DNA Release Meizhen Yin,† Ke Ding,† Radu A. Gropeanu,† Jie Shen,‡ Ru¨diger Berger,† Tanja Weil,*,† and Klaus Mu¨llen*,† Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany, Department of Entomology, Institute of Agronomy and Biotechnology, China Agricultural University, 10094 Beijing, China Received July 17, 2008; Revised Manuscript Received September 24, 2008

Water-soluble core-shell star polymers consisting of a dendritic polyphenylene core and an outer shell containing a defined number of amino groups have been synthesized via atom transfer radical polymerization (ATRP). All macromolecules efficiently interacted with a diverse set of DNA fragments, and stable complexes were formed and visualized by atomic force microscopy. The observed tight binding of DNA, which was found in the subnanomolar range, was mainly attributed to strong electrostatic interactions. Complex stoichiometries between the polyelectrolytes were controlled via the number of amino groups of the star polymers, and well-defined nanoscopic architectures were formed. DNA was released from the complexes after treatment with high concentrations of sodium chloride in aqueous solution. Such star polymers, which allow the binding and release of DNA, represent attractive candidates for the development of novel anion-exchange resins for DNA purification or as nonviral vector systems for gene delivery.

Introduction The design and controlled fabrication of core-shell nanoparticles consisting of inorganic or organic core molecules coated with hydrophilic polymer shells represents a field of emerging interest in polymer and pharmaceutic research.1 Their applications are broad, ranging from catalysis, water-borne coatings, and bioseparations to controlled release of drugs and gene therapy.2,3 Representative materials include polymeric micelles based on di- or multiblock copolymers,4 dendritic micelles,5 or inorganic nanoparticles coated with polymer shells.6,7 Conventional polymeric micelles represent thermodynamic aggregates of amphiphilic molecules above their critical micelle concentration. In such cases, when the concentration of a polymeric micelle drops below the critical micelle concentration, its micellar structure becomes unstable and dissociates into free chains.8 In contrast to those conventional micellar systems, dendritic core-shell macromolecules form unimolecular micelles in which the hydrophilic and hydrophobic segments are connected covalently.5 Therefore, their micellar structure is static rather than dynamic, and it is maintained at all concentrations and in a variety of solvents thus offering structural stability combined with an access to low nanometer sizes, monodispersity, solubility, unique spherical shapes, and the availability of a defined number of functional groups at the surface.9-12 In the past, dendritic core-shell macromolecules have successfully been applied as hosts for the noncovalent uptake of small guest molecules into the dendritic branches (“dendritic box”).13-15 In addition, the interaction of structurally defined PAMAM dendrimers and DNA has been studied previously.16 However, the uptake and release of DNA by a dendritic core-shell macromolecule is still elusive. To date, viral carriers are mainly used for the delivery of genetic material, although their application is usually restricted to single uses * Corresponding author. E-mail: [email protected] (T.W.); [email protected] (K.M.). † Max Planck Institute for Polymer Research. ‡ China Agricultural University.

since they might elicit immune responses.17 There have been several attempts to design improved, nonviral DNA delivery agents.18 However, despite nearly two decades of research, nonviral gene delivery systems are still limited by poor transfection efficiencies.18 This manuscript deals with our research toward amphiphilic core-shell macromolecules capable of complexing and releasing DNA as a selective response to an ion gradient. Recently, we have focused on the synthesis of functionalized polyphenylene dendrimers coated with oligolysine groups by applying peptidesubstituted cyclopentadienones or via a functionalization of an N-alkyl maleimide-substituted dendrimer with a thiol-endcapped peptide.19-21 The presence of a stiff, hydrophobic core of distinct size and geometry leads to the formation of defined nanoscopic sizes and shapes in solution. In addition, fine-tuning of the number of amino groups within the polymer shell should allow a manipulation of complex stoichiometries all together, leading to the formation of defined supramolecular architectures. Functionalized stiff polyphenylene dendrimers coated with precise numbers of initiating groups were successfully employed as macroinitiators for the construction of star polymers via atomic transfer radical polymerization (ATRP).22 However, the synthetic routes toward polyphenylene macroinitiators were quite tedious,22 and therefore an optimization of the synthesis procedures toward higher generation polyphenylenes was required. Via ATRP, different core-shell macromolecules were generated bearing a varying number of positive charges at the outer shell. Structurally similar macromolecules containing several free amino groups have shown to enable a fast cell uptake, which makes them attractive for drug delivery applications.23 Recently, biological applications on animal cells with structurally similar core-shell macromolecules containing multiple -COOH or -NH2 groups revealed specific histochemical labeling of the cell nucleus24 or extracellular matrix.25 Here, complex formation and release of DNA were investigated as a function of the number of amino groups and the salt gradient. The combination of strong DNA complex

10.1021/bm800797j CCC: $40.75  2008 American Chemical Society Published on Web 10/22/2008

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Figure 1. Two-dimensional (2D) structures of first-, second- and third-generation polyphenylene dendrimer macroinitiators (M1, M2, M3) and protected or deprotected star polymers (P1, P2, P3, P4, P5, P6) with 6, 12, and 24 polymer chains.

formation and DNA release at defined conditions paves the way toward potential applications as novel anion-exchange resins for DNA purification as well as nonviral vector systems for gene delivery applications.

Experimental Section 1. Reagents. 1,3,5-Triethynylbenzene (Avocado, 98%), CuBr (Aldrich, 99.999%), 4,4′-di-tert-butyl-2,2′-bipyridine (DTB-bipy) (Aldrich, 98%), phenol (ARCOS, 99%), trimethylsilyl chloride (1 M in dichloromethane), and 2-butanone (ARCOS, 99%) were used as purchased. 3-(4-Methoxyphenyl)-2,4,5-triphenylcyclopenta-2,4-dienone (CP-OMe) and 3,4-bis-(4-methoxyphenyl)-2,5-diphenylcyclopenta-2,4-dienone (CP(OMe)2) were synthesized according to published procedures.26,27 2-tert-Butoxycarbonylaminoethyl methacrylate (Boc-AEMA) was synthesized according to previous publications.28,29 The synthesis of first- and second-generation functionalized dendrimers (M1 and M2, Figure 1) with initiator groups at the surface have been described previously.22 Polyphenylene dendrimers containing ethynyl groups (2, Supporting Information Scheme 2) were synthesized via divergent synthesis procedures.30 2. Instruments. Gel permeation chromatography (GPC) analyses were performed in (i) tetrahydrofuran (THF) with a Waters pump model 515, detectors RI 101 ERC and UV-vis S-3702 Soma (255 nm), temperature: 30 °C, standards: PSt. 1H NMR spectra were recorded on a “Bruker-Spectrospin” instrument (250 MHz) at room temperature. 13 C NMR spectroscopy was performed on a “Bruker AMX 300” spectrometer at room temperature. Dynamic light scattering (DLS) measurements were performed on an ALV 5000 Correlator (Malvern Instruments), ALV-SP81 goniometer, Avalanche photodiode laser krypton-ion-laser 647.1 nm, Spectra Physics model Kr 2025. Matrix-

assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry measurements were performed on VG ZAB2-SE-FPD Spectrofield, Bruker Reflex I (MALDI-TOF) and Bruker Reflex II (MALDI-TOF) mass spectrometers. 3. General Procedure for Radical Polymerization by ATRP. The monomer was placed in a Schlenk flask and dissolved in the 2-butanone. CuBr was then added, and the mixture was degassed by three freeze-pump-thaw cycles. The ligand DTB-bipy was added under argon atmosphere. Then, the solution was stirred at 25 °C for 10 min. Finally, the initiator was added, and the flask was sealed and placed in a thermostatic oil bath at 70 °C to start the reaction. Cooling with liquid nitrogen after a defined reaction time stopped the polymerization reaction. The monomer conversion was determined by 1H NMR spectroscopy. The reaction mixture was diluted and eluted through a column filled with neutral alumina to remove the copper complex. Thereafter, the solvent was removed under reduced pressure, and the polymer was isolated by precipitation from methanol and drying under vacuum to constant weight. 4. Hydrolysis of tert-Butoxycarbonyl (BOC) in Core-Shell Nanoparticles. The core-shell star polymer of Boc-AEMA (100 mg) was dissolved in dichloromethane (30 mL) in a 100-mL round-bottomed flask. Phenol (3 M in dichloromethane, 5 mL) and chlorotrimethylsilane (1 M in dichloromethane, 5 mL) were added, and the mixture was stirred for 2 h at room temperature. The solution was evaporated to dryness, and the residue was dissolved in methanol (10 mL). Then, the product was precipitated upon addition of diethyl ether, and the solid was filtered and dried under reduced pressure. 5. Isothermal Titration Calorimetry Measurements. An isothermal titration calorimeter (ITC, MicroCal Inc., Northampton, MA) was used in order to assess the binding constant of the star polymers and DNA. The ITC instrument was periodically calibrated electrically with

Dendritic Star Polymers for DNA Binding & Release an internal electric heater. In the microcalorimetric titration, typically, a constant 5 µL portion of the star polymer solution (11 µM for P2, 7.5 µM of P4, and 2 µM of P6 in 50 mM Tris · HCl buffer (pH 7.4)) was successively injected 54 times into the reaction cell (1.4078 mL) filled with a 13.1 nM (22.5 µg/ml) pUC19 plasmid DNA solution in Tris · HCl buffer. The time gap between two consecutive injections was 5 min, which is required to allow the system to reach the equilibrium after each injection. The dilution of the star polymer solutions upon addition to the pure buffer solution placed in the cell was also determined, using the same number of injections and the same concentrations as employed in the titration experiment. The heats of interaction during each injection were measured by integration of each titration peak using the ORIGIN 7 software (OriginLab Co. Northampton, MA, delivered with the VP-ITC). For each injection, the dilution heats determined in the control experiment were subtracted from the heats obtained in the interaction experiment. The resulting corrected heats of the interaction curves were used for the calculations of the stoichiometry, molar enthalpy, equilibrium constant, entropy, and Gibbs free energy of the complexation. The number of the positively charged residues of the star polymers that participate in polyplex formation with DNA was determined considering that, at neutral pH, all of the phosphate residues of plasmid DNA pUC19 are negatively charged and all are involved in complexation (2686 bp, 5372 negative charges). The exact knowledge of the number of phosphate residues of one plasmid DNA molecule and the determined stoichiometry allows the estimation of the number of the amino groups of the core-shell macromolecules participating in polyplexe formation, under the assumption that each phosphate residue interacts with one amino group of the core-shell macromolecules. 6. AFM Sample Preparation. P2 was completely dissolved in water and then diluted in buffer solution (10 mM Tris-HCl pH 7.4, 1 mM NiCl2) to 1.25 mg/L. For the complexation reaction, P2 and DNA were mixed at a charge ratio of 2.17:1 (number of negative charges of DNA sample versus the number of positive charges of the star polymer) and diluted in buffer. A drop of 20 µL sample solution was deposited on freshly cleaved mica (Plano GmbH, Germany) and left to incubate for 5 min. Then, the surface was washed with 200 µL of buffer solution and mounted to the sample stage (USA). Imaging was performed in tapping mode in liquids (E-scanner, Multimode, Nanoscope IIIa controller Veeco Instruments, San Clemente, CA). Oxide-sharpened silicon nitride cantilevers (NP-S, Veeco Instruments; 115 µm long, 17 µm wide, 0.6 µnm thick) with an integrated tip (nominal spring constant of 0.32 N/m) were applied. A driving frequency between 8 and 10 kHz for imaging in buffer solution was selected. The images were recorded with a scan size of 1 × 1 µm2 at a scan rate of 1 Hz and by adjusting soft tapping parameters. Raw topography data were modified by applying the first-order “flatten” filter. A stoichiometry of 1:1 was used for complex formation. 7. Agarose Gel Electrophoresis. A 0.8% agarose (w/v) gel in 1X Tris-Borate-EDTA (TBE) buffer was performed for 2 h at 4 V/cm. A 5 µL portion of 1 kb DNA ladder (PeqLab) was incubated with 5 µL of 0.2% core-shell macromolecules for 10 min before loading. The gel was stained with 1 µg/mL ethidium bromide in 1X TBE for 30 min after electrophoresis. The photograph was taken under UV light (wavelength 365 nm).

Results and Discussion Synthesis of the Functionalized Water-Soluble Star Polymer. The characterization and the formation of well-defined core-shell architectures based on first- and second-generation polyphenylene macroinitiators has already been investigated in detail.22 In the present study, a third-generation polyphenylene dendrimer substituted with 24 2-bromo-2-methylpropionic ester groups at the periphery was prepared via an optimized procedure. A detailed synthetic procedure including the application of symmetric and asymmetric cyclopentadienons and the

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Table 1. Characterization of Star Polymers P1, P3 and P5, Which Were Obtained via an ATRP Reaction of Boc-AEMA at 70°C (M/Br-I/CuBr/DTB-bipy ) 200:1:1:1) polymer

initiator a

no.

DLS Rh [nm]

no.

DLSa Rh [nm]

Mtheor [g/mol]

MNMR [g/mol]

Mn,GPC [g/mol]

PDb

P1 P3 P5

3.4 4.9 8.2

M1 M2 M3

1.522 2.2

15 949 32 962 66 988

18 600 38 400 71 000

14 000 27 000 51 000

1.19 1.18 1.16

a DLS experiments were performed in dichloromethane. for polydispersity.

b

PD stands

characterization of all products is described in the Supporting Information (Schemes S1 and S2). By applying the first-, second-, and third-generation functionalized dendrimers (M1, M2, M3, Figure 1) as macroinitiators, different generations of core-shell macromolecules were prepared by ATRP polymerization of Boc-AEMA. The general synthetic strategy is depicted in the Supporting Information (Scheme S3). It is worth noting that the polymerization conversion needs to be kept below 20% in order to avoid intermolecular interactions and to facilitate an easy monitoring of the chain growth by 1H NMR spectroscopy. The ATRP data of Boc-AEMA is summarized in Table 1. Three different techniques were applied in order to estimate the molecular weights of these core-shell macromolecules: The theoretical molecular weights (Mtheor, Table 1) were calculated by the conversion of the polymerization reaction. In addition, the molecular weights based on NMR experiments, MNMR, were calculated by comparing the relative signal intensities of the aromatic protons of the dendritic initiators at 7.3-6.1 ppm with those of the methylene protons present in the Boc-AEMA side chains at 4.0-3.5 ppm. Furthermore, molecular weights were obtained after GPC experiments which provided the Mn,GPC results listed in Table 1. In accordance with previous experiments, the theoretical molecular weights (Mtheor) and the calculated molecular weights from NMR experiments (MNMR) were found to be higher than the Mn,GPC values obtained by GPC.22 The repeating units of the star polymers were controlled by the monomer conversion, which was monitored via 1H NMR spectroscopy. Monomodal distributions were observed in all cases together with narrow dispersities. Table 1 reveals that the molecular weights of the star polymers increased with the dendrimer generation of the star polymer. An additional important characteristic of these core-shell star polymers is their size, obtained as the hydrodynamic radius (Rh) in the given solvent. The Rh values were measured by DLS in dichloromethane (7.0 g/L, Table 1). All star polymers (Rh about 3-8 nm) were considerably larger than their respective macroinitiators (Table 1),22 which further supports the formation of core-shell architectures. An increase of the generation of the polyphenylene dendrimer resulted in an increase of the hydrodynamic radius of the macromolecule. Interestingly, P5 was found to be significantly larger than P3 and P1, which could be attributed to the formation of a globular shape of P5 due to the presence of a large number of surface groups leading to the more extended polymer chains by preventing their back-folding. Water-soluble star polymers were achieved after the removal of the BOC protective groups by treatment with phenol and chlorotrimethyl silane (Scheme S3).31 All core-shell macromolecules with free amino groups revealed high water solubility (>10 g/L), which is crucial for investigations in a biological environment. For the sake of clarity, the abbreviation G1(10)6 in Figure 1 describes a core-shell macromolecule containing

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Figure 2. (A) DLS data (Rh in Tris buffer, pH ) 7.4) of the final water-soluble core-shell macromolecules (P2, P4, P6). (B) DLS data (Rh in Tris buffer, pH ) 7.4) of P2, plasmid DNA pUC19 and pUC19/ P2 at a ratio of 1:1.

a first-generation dendrimer core with six arms and 10 repeating units of each polymer chain. The sizes of the water-soluble star polymers (Figure 2A, P2, P4, P6) in buffer solution were determined in DLS experiments, and slightly larger Rh values were found compared with their protected analogues (Table 1, P1, P3, P5) dissolved in dichloromethane. This observation could be attributed to electrostatic repulsion between those polymer chains bearing free amino groups, thus inducing the formation of more extended shapes in solution. Furthermore, the Rh values of the particles do not show significant variations at different angles, indicating the existence of isolated, welldefined macromolecules. Formation of DNA/Star Polymer Complexes. All star polymers P2, P4, and P6 bear free primary amino groups, which are protonated under acidic or physiologic conditions. Therefore, their interaction with negatively charged macromolecules such as DNA could occur through electrostatic forces in a neutral buffer solution. In order to assess whether differently sized DNA fragments interact with these star polymers, a smart DNA ladder containing 14 series of DNA fragments (sizes ranging from 250 to 10 000 base pairs) was selected for complex formation experiments. Here, the DNA ladder was incubated with star polymers P2, P4, and P6 at a molar ratio of 1:1 of negatively charged DNA and positively charged star polymers for 15 min. These mixtures were then loaded onto a 0.5% agarose gel for an electrophoresis experiment. Free DNA fragments moved through the gel and separated from each other depending on their sizes, while P2, P4, and P6 star polymers effectively neutralized DNA and formed stable DNA complexes, which did not move in the gel. All complexed DNA fragments remained at their original (start) position in the wells (Figure 3), indicating that the star polymers strongly interacted with all DNA fragments ranging from 250 to 10 000 bp, a size range covering most of the DNA sizes used for gene engineering applications. The formation of DNA/star polymer complexes was further demonstrated by DLS experiments in buffer solution, as shown in Figure 2B. Here, plasmid DNA pUC19 was used for complex formation and P2/pUC19 complexes of similar sizes were found with average hydrodynamic radii (Rh) of 85 nm, which differed significantly compared with that of isolated P2 (Rh ) 4 nm) or free pUC19 (Rh ) 40.6 nm). Estimation of Complex Stabilities. The binding affinities and the thermodynamic data of the interaction of core-shell macromolecules P2, P4, and P6 with pUC19 were investigated by applying isothermal titration microcalorimetry (ITC). It is worth mentioning that the molecular weights of P2, P4, and P6 used for ITC experiments are the theoretically calculated ones. ITC was previously used for the thermodynamic characterization of polyplex formation,32-34 and it allows the acquisition of thermodynamic data in one experiment with high

Figure 3. Gel electrophoresis of free DNA ladder and DNA ladder complexes with star polymers P2, P4, and P6. The arrow indicates the position on the wells where the samples were loaded (start). All three polymer/DNA complexes were not able to move through the gel.

precision and without the necessity to apply a radio-labeled ligand.35 During the titration of DNA with small amounts of a polycation, the degree of charge neutralization of the DNA increases with the increase of the polycation concentration. Close to charge neutralization, DNA condenses into a compact structure, which often precipitates from solution.36 In order to determine the thermodynamic parameters, we have assumed that the complexation is of noncooperative nature and is independent of the pDNA nucleotide sequence.37 In the past, noncooperativity has been reported in the case of oligo-lysine/DNA complexation studies,38 and we have applied this simplification because of the similarity of the polylysine oligomers with the amino-containing chains of the shell of P2, P4, and P6 nanoparticles. In addition, the good correlation of the observed and calculated data also points to a noncooperativity. As variables, the enthalpy (∆H), the binding constant (KB), and the stoichiometry (n) were considered. Herein, the formation of polyplexes of positively charged star polymers P2, P4, and P6 and pUC19 was studied at 25 °C.

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Table 2. Thermodynamic Data of the Interaction of Cationic Star Polymers with Plasmid pUC19 DNA a

macro-molecule

N

P2 P4 P6

97.5 ( 0.4 45.9 ( 0.2 30.0 ( 0.1

N*

theor. no. of amino groups

∆Hobs [kcal/mol]

∆Sobs [kcal/molK]

-T∆Sobs [kcal/mol]

∆Gobs [kcal/mol]

KB,obs × 109 [l · mol-1]

KD,obs [nM]

55 ( 0.3 116 ( 0.5 196.7 ( 2

60 120 240

71.1 ( 0.5 85.6 ( 0.6 106.1 ( 1.9

277 330 398

-82.6 -98.3 -118.6

-11.5 ( 0.2 -12.7 ( 0.1 -12.5 ( 0.1

0.3 ( 0.04 1.9 ( 0.4 1.8 ( 2.02

3.1 ( 0.4 0.5 ( 0.1 0.55 ( 0.09

b

a N stands for the complex stoichiometry; b N* corresponds to the approximate number of positive charges interacting with one molecule pUC19 under the assumption that neutral complexes were formed.

Figure 4. Titration of 8.7 nM plasmid DNA pUC19 with (a) 11.4 µM P2 and (b) 3.2 µM P6. Conditions: 25 °C, 50 mM Tris buffer pH 7.4. The upper panels reveal the raw titration data, the enthalpy, entropic factor, and the Gibbs energy of the interactions. In the lower panels, the integrated heats and the calculated thermodynamic data are shown. The red line represents the best fitting obtained using a one-binding-site model.

The acquired thermodynamic parameters of this interaction are listed in Table 2, and the titration curves given in Figure 4A,B reveal a good correlation between the fitted curves and the experimental data. Interestingly, very high affinities in the nano- and subnanomolar range of the star polymers P2, P4, and P6 for DNA were found. The measured binding constants (KB of P2/pUC19, P4/pUC19, and P6/pUC19 complexes equals 3 × 108, 1.9 × 109 and 1.8 × 109 L · mol-1, respectively) were significantly higher than those reported for linear polycations (KB ) 104-107 L · mol-1).39,40 Surprisingly, the dissociation constant (KD) of P4/pUC19 complexes decreased by a factor of about 6 compared to P2/pUC19 complexes, indicating tighter interactions between P4 and pUC19. However, a further increase of the number of amino groups (P6) did not lead to significant changes of the binding constant (Table 2). Stoichiometry of Star Polymer/pUC19 Complexes. Polyplex formation of pUC19 and the cationic core-shell macromolecules occurred with positive enthalpies ∆H of 71.1 kcal/ mol (P2), 85.6 kcal/mol (P4), and 106.1 kcal/mol (P6). This positive enthalpy was compensated by a higher entropy term ∆S, which could be attributed to the release of water molecules and counterions from the contact surfaces of both macromolecules into the bulk solvent.41 Both the entropy and the enthalpy terms were found to increase with increasing dendrimer generation (Table 2, Figure 5). Interestingly, considering the growth of the “theoretical” number of amino groups (Table 2) of each star polymer by a factor of 2 (P2 versus P4, P4 versus P6) and 4 (P2 versus P6), the relative contribution of each amino group to the entropy or enthalpy terms was obviously reduced in higher dendrimer generations. In addition, a clear correlation between

Figure 5. Representation of the enthalpy (red), entropy (green), and Gibbs energy (blue) of the interaction of P2, P4, and P6 dendrimers with plasmid DNA pUC19 (50 mM Tris · HCl pH 7.4, 25 °C).

the number of amino groups within the polymer shell of the star polymers and the resulting composition of the complexes (N, Table 2) at the thermodynamic equilibrium was found. A total of 97.5 molecules of P2 were found to interact with one molecule of pUC19, whereas P4/pUC19 and P6/pUC19 complexes consisted of 45.9 and 30 star polymers per molecule of pUC19 on average (Table 2). Obviously, increasing the numbers of amino groups within the polymer shell facilitated the formation of complexes with a higher DNA content and thus with a reduced number of star polymers. Therefore, a manipulation of complex stoichiometries could be achieved by modulating the number of amino groups within the dendritic shell. Estimation of the Number of Primary Amino Groups of Star Polymers P2, P4, P6. The average number of amino groups of each star polymer interacting with one molecule pUC19 (N*, Table 2) was assessed from the number of negative charges of pUC19, the complex stoichiometries obtained in the ITC experiments described above, and under the assumption

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Figure 7. Gel electrophoresis of 4.1 Kb plasmid DNA binding by polymers and releasing under high NaCl concentration. The arrow indicates the position on the wells where the samples were loaded initially.

Figure 6. AFM images of P2 particles (A), free DNA (B), and complex of DNA/P2 in buffer (C). (A) P2 particles distribute either in aggregated or scattered forms in the buffer. (B) DNA fragments are in free shapes in buffer. (C) P2 particles bind to DNA and knot it in buffer solution.

that all phosphate groups of DNA were neutralized.42 Table 2 displays the calculated number of amino groups “N*” as well as the “theoretical” number of amino groups under the assumption that each initiator has started the growth of a polymer chain with the adjusted chain lengths, e.g., 10 amino groups for each polymer branch, and six branches for the first- and 12 branches for the second-generation star polymers. A good correlation between N* and the theoretical number of amino groups was obtained for P2 and P4. However, considering the thirdgeneration star polymer P6, a significantly reduced number of amino groups compared with the theoretical values calculated by the conversion of the polymerization reaction was gained. On one hand, this result might be attributed to the fact that P6 essentially bears less amino groups than calculated. On the other hand, the formation of positively charged complexes due to a lower number of amino groups that participate from each core-shell dendrimer in the interaction with pUC19 could have as consequence an underestimation of the real number of amino groups by ITC. However, according to these results, ITC experiments provide an alternative method for the estimation of the number of amino groups of these star polymers, which is of particular importance since alternative characterization methods such as GPC were not applicable for these watersoluble star polymers. Star Polymer/DNA Complexes on Surfaces. Subsequently, the microstructures of the complexes of P2 and DNA were studied by atomic force microscopy (AFM). AFM is widely used to investigate the micromorphology in biological and polymer systems.43-45 AFM image revealed the distribution of P2 as either a single molecule or aggregated assemblies on a mica surface (Figure 6A). In addition, 2.2 Kb linear DNA fragments were deposited on a surface (Figure 6B). The morphology of free DNA was found to be smooth, of flexural shape, and thus similar to published AFM images.46,47 In order to investigate the star polymer/DNA assemblies, P2/DNA complexes were formed by mixing both macromolecules in a neutral buffer solution (see Experimental Section) and subse-

quent deposition on a mica surface. P2 macromolecules interacted with DNA at multiple sites and knotted the flexural DNA at the interaction points into semicondensed complexes (Figure 6C). Similar structures have been observed in previous work.47-49 Here, an excess of DNA was applied, which might lead to the formation of complexes still bearing free DNA. Obviously, the presence of highly cationic star polymers leads to the formation of knots consisting of P2/DNA at central positions encircled by free DNA. Therefore, Figure 6C could be interpreted as a transition state before condensed DNA/ complexes are formed upon continued addition of polycations. Obviously, complex formation starts at a few sites only where free DNA chains interact with several star polymers, thus forming dense knots. Then, depending on the number of available star polymer molecules and on the number of amino groups, DNA chains fold around those knots leading to the formation of dense DNA/star polymer complexes. Decomplexation of Plasmid DNA. Since plasmid technology is a common tool in molecular biology for gene cloning experiments, a 4.1 Kb plasmid DNA (pOT2) was selected to investigate whether star polymers P2, P4, and P6 could be applied as resins for the isolation or purification of plasmid DNA via first a binding and then a releasing step. pOT2 was mixed with each of the three star polymers (at 1:1 molar ratio of charges), respectively. Then, the salt concentration (NaCl) was elevated to 1 M and 2 M. Consistent with the results shown in Figure 5, all star polymers first interacted with pOT2 (Figure 7). After increasing the ion strength in the pOT2/star polymer complex solutions to 1 M NaCl, the majority of the DNA molecules were released from the complexes of P2/DNA and P4/DNA, but less release occurred for P6/DNA complex. Upon further increasing the NaCl concentration to 2 M, the majority of the DNA molecules were released from the complexes of P6/DNA. Here, very few pOT2 complexes remained at the “start” position in the wells (Figure 7). A similar path length of released pOT2 versus free pOT2 was observed, indicating that the DNA was still intact and not damaged during the complexation reaction and the presence of high salt concentrations. Interestingly, the remaining amount star polymer/DNA complexes visualized by the emission of the DNA intercalator ethidium bromide after salt addition seemed to increase for P4 and P6, which might reflect the lower dissociation constant KD of these complexes (Table 2) compared to P2/pUC19 complexes. The effect of polymer ionization on DNA decomplexation has successfully been studied via ITC previously.32 Here, P2/ pUC19 was used to assess the influence of sodium chloride on complex formation. In extension of the titration experiments of Figure 4A where no salt was applied, three additional measurements were performed applying increasing salt concentrations

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Table 3. Thermodynamic Parameters of the Interaction between P2 and pUC19 DNA in 50 mM Tris · HCl Buffer (pH 7.4) and Different NaCl Concentrations P2/ pUC19

N*a

∆Hobs [kcal/mol]

∆Sobs [cal/mol · K]

-T∆Sobs [kcal/mol]

∆Gobs [kcal/mol]

KB,obs [mol · lL-1] × 108

KD,obs [mol · L-1] × 10-9

0 mM NaCl 50 mM NaCl 100 mM NaCl 1 M NaCl

97.5 ( 0.4 95.6 ( 0.6 85.0 ( 0.6

71.1 ( 0.5 50.7 ( 0.6 39.0 ( 0.6

277 208 170

-82.6 -62.0 -50.7

-11.5 ( 0.2 -11.3 ( 0.3 -11.7 ( 0.5

3.2 ( 0.4 2.4 ( 0.4 3.7 ( 0.6

3.1 ( 0.4 4.2 ( 0.8 2.7 ( 0.5

a

Stoichiometry, e.g., number of P2 molecules interacting with pUC19.

Figure 8. ITC curves for the complex formation between P2 and pUC19 DNA in different NaCl concentrations.

(with 50 mM, 100 mM and 1 M sodium chloride, Table 3). With increasing ionic strength, a reduction of all thermodynamic parameters was observed, and, after application of a high sodium chloride concentration (1 M), complex formation was no longer detected by ITC (Figure 8). This observation supports the result of DNA releasing under higher NaCl concentration in gel electrophoresis experiments. Interestingly, within the experimental errors of the ITC experiment, no significant changes of the binding constants KB (2.4-3.7 × 108 L · mol-1) of the complexes were found up to 100 mM sodium chloride, and the ratio of P2 molecules in the complexes only slightly decreased, thus indicating a sufficient complex stability under these conditions. These results could be advantageous for cell transfection experiments since stable complexes are formed under physiological conditions, even at high ion strengths in which DNA is protected from proteolytic degradation.41,50

Conclusion An optimized synthetic strategy facilitated the preparation of dendritic macroinitiators in an economic way, thus allowing considerably shorter reaction times and only small amounts of the reagents. By applying this improved procedure, three generations of water-soluble star polymers were synthesized in a controlled way via ATRP. All water-soluble and nanosized (3-8 nm) macromolecules reported herein efficiently interacted with a diverse set of DNA fragments and two different kinds of plasmid DNA. The observed tight binding to DNA, which was found in the nano- and sub-nanomolar range, was attributed to strong electrostatic interactions due to high charge densities of the dendritic shells. Complex stoichiometries between both polyelectrolytes were controlled via the number of amino groups of the star polymers via ATRP, leading to the formation of welldefined nanoscopic architectures. A large number of amino groups within the polymer shell facilitated the formation of complexes with a higher DNA content and thus with a reduced number of star polymers. These results open up the possibility to investigate the impact of complex stoichiometries on gene transfection, which might improve our understanding of such complex processes. Star polymer/DNA complex formation was

visualized by AFM and facilitated a hypothesis about the process of complex formation as well as the connected morphology changes. All polyelectrolyte complexes consisting of different star polymers revealed a high thermodynamic stability at elevated salt concentrations since nearly no changes of the binding constants and complex stoichiometries were observed. However, DNA release was achieved as a response to a further increased ion gradient. Because of their ease of synthesis, the unique possibility to adjust the number of charges via ATRP and their capabilities of capturing and releasing different kinds of DNA, these star polymers represent attractive research tools to further explore gene transfection. Additionally, they could serve as novel anionexchange resins for the purification of DNA since in principle, high quantities of DNA could be captured. Their potential application as novel nonviral vector systems for gene delivery is under current investigation, and the first in vitro experiments including cytotoxicity tests are ongoing. Future work will focus on in vitro purification of DNA from lysate of cells or tissues and on in vivo gene delivering into live cells. Acknowledgment. Funding from the Deutsche Forschungsgemeinschaft (SFB 625, “From Single Molecules to Nanoscopically Structured Materials”) is gratefully acknowledged. We acknowledge Hans Jürgen Butt for continuous fruitful discussions and support; we also thank Christine Rosenauer for DLS measurements and Don Cho for comments on the manuscript. Supporting Information Available. Synthesis and characterization of symmetric and asymmetric cyclopentadienons and the third generation of functionalized polyphenlene dendrimer, as well as a general synthetic strategy to polymer (Schemes S1-S3). This material is available free of charge via the Internet at http://pubs.acs.org.

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