Complexation of Linear DNA and Poly(styrenesulfonate) with Cationic

Article pubs.acs.org/JPCB

Complexation of Linear DNA and Poly(styrenesulfonate) with Cationic Copolymer Micelles: Effect of Polyanion Flexibility Yaming Jiang,† Dustin Sprouse,‡ Jennifer E. Laaser,‡ Yogesh Dhande,† Theresa M. Reineke,*,‡ and Timothy P. Lodge*,†,‡ †

Department of Chemical Engineering & Materials Science, University of Minnesota, 421 Washington Ave. SE, Minneapolis, Minnesota 55455, United States ‡ Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, Minnesota 55455, United States S Supporting Information *

ABSTRACT: The complexation of linear double stranded DNA and poly(styrenesulfonate) (PSS) with cationic poly(dimethylamino ethyl methacrylate)-block-poly(n-butyl methacrylate) micelles was compared in aqueous solutions at various pH values and ionic strengths. The complexation process was monitored by turbidimetric titration, as a function of the ratio (N/P) of amine groups in the micelle corona to the number of phosphates (or sulfonates) in the polyanion. The size, structure and stability of the resulting micelleplexes were studied by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM). In the short chain regime, where the contour lengths of the polyanions are shorter than or comparable to the micelle corona thickness, micelleplexes with DNA oligomers show very similar behavior to complexes with short PSS chains, in terms of titration curves and structural evolution of the complexes as a function of charge ratio. However, in the long chain regime, where the contour length of the polyanion far exceeds the micelle radius, micelleplexes of linear DNA show titration curves shifted toward lower N/P ratios, reduced stability at N/P < 1, and a higher percentage of small complexes at N/P > 1 compared to complexes with long chain PSS. Furthermore, at 1 M ionic strength, the cationic micelles could still complex with long chain PSS, but not with DNA of the same total charge. These differences are attributed to the flexibility difference between the polyanion chains, and possible mechanisms are proposed. This work highlights the importance of chain flexibility in complexation of dissimilar polyelectrolyte pairs, a factor that could therefore help guide the future design of micelleplexes for various applications.

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formation22 and the chemistry and architecture of the micelle36 or the linear polyelectrolyte.37,38 Therefore, incorporation of polyelectrolytes of greater structural hierarchy, such as polyionic micelles, could potentially promote the general utility of the such complexes. One promising application is to construct polyelectrolyte complexes with tunable physical properties for nucleic acid delivery.39−41 Cationic polymer vehicles can associate with nucleic acids to form interpolyelectrolyte complexes that are stabilized against aggregation or degradation, and that promote higher delivery efficiency than uncomplexed polynucleotides.42 Genetic payloads have the ability to treat intractable therapeutic targets including many acquired and inherited diseases, such as cancer, HIV, and muscular dystrophy.43−45 Most polymeric vehicles developed to date are linear or branched polymers that complex with DNA to form polyplexes.40,46 However, polyplexes suffer drawbacks such as poor control of structure, ill-defined composition, and lack of understanding of their solution behavior, which potentially hinders clinical develop-

INTRODUCTION Polyelectrolyte complexation is largely an entropy-driven process whereby oppositely charged polyelectrolytes release their counterions, and associate by electrostatic interaction to form complexes.1,2 These polyelectrolyte complexes, either in solution or solid state, are versatile materials that are utilized in a diverse array of applications, such as water treatment,3,4 encapsulation,5,6 filtration,7,8 self-healing materials,9,10 and drug delivery.11,12 Most studies to date have focused on complexes of linear polyelectrolytes,13−16 but more elaborate architectures have been introduced that not only expand the array of possible structures,17−21 but also offer additional routes to tune their properties.22−25 One particular example is the introduction of polyionic micelles in polyelectrolyte complexation.18,19,26,27 Polyionic micelles are assemblies of amphiphilic block polymers with one ionic block and one hydrophobic block, and their solution properties have been thoroughly studied.28−32 Compared to linear−linear polyelectrolyte complexes, linearmicellar complexes or “micelleplexes” show structures ranging from single-micelle onion-like structures27,33,34 to aggregates containing multiple micelles bridged by linear polyelectrolytes.25,35 Micelleplex structures and properties can be controlled by additional factors, such as the pathway of © 2017 American Chemical Society

Received: April 21, 2017 Revised: June 14, 2017 Published: June 30, 2017 6708

DOI: 10.1021/acs.jpcb.7b03732 J. Phys. Chem. B 2017, 121, 6708−6720

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The Journal of Physical Chemistry B ment and application.41,45 One alternative is to replace linear polycations with polycations of predefined structure, such as cationic micelles, to complex with DNA and form micelleplexes.35,47−49 As the resulting structure builds on the preassembled micelles, the composition and properties of micelleplexes are potentially easier to control. In addition, polymeric micelles have been widely explored as carriers for small-molecule drugs,50−52 and thus cationic micelles could serve as dual-functional vehicles that deliver both gene and drug payloads simultaneously. 53,54 Although the concept of micelleplexes is not new, there have been few studies on this topic, most of which focused on biological performance.35,47−49 To utilize the full potential of micelleplexes, further characterization of their solution properties is needed to elucidate the physical principles that govern their complexation behavior, and thereby build a foundation for achieving control and tunability of their physical properties. A diblock copolymer cationic micelle with a relatively simple structure is suitable for such physical studies, and the obtained insights can guide the future design of polymeric vehicles with potentially more elaborate structures.25,36 DNA is a semiflexible polyelectrolyte where the stiffness originates from the double helical structure.55 This intrinsic stiffness will likely influence how DNA complexes with cationic micelles, especially considering the significant disparities in shape and charge density between DNA and polymeric micelles. The influence of chain flexibility has not been investigated experimentally in the context of complexation to polymeric micelles, but previous studies with surfactant micelles and proteins have indicated that chain stiffness is an important factor. For example, with small molecule surfactant micelles, the stiffness of the linear polyelectrolyte was found to reduce its binding strength to the oppositely charged micelles.56 Simulations of semiflexible polyelectrolyte chains interacting with oppositely charged spheres revealed that stiffness affects the extent of complexation,57 as well as the structure of the complexes.57−60 However, these studies almost exclusively investigated charged hard spheres, and did not capture the structural evolution of the complexes. Previous studies of micelleplexes, either with oligonucleotides61−63 or plasmids,35,48 have not compared the behavior of stiff polyanions with flexible ones. Although many studies have explored the properties of complexes composed of polymeric micelles and oppositely charged flexible polyelectrolytes,25,27,33,34,36 insight into the design of polymeric micelle-DNA complex was limited as the potentially critical influence of DNA chain stiffness is unknown. In this work, we compare the complexation of polymeric cationic micelles with linear double stranded DNA (dsDNA) and flexible poly(styrenesulfonate) (PSS) with a similar total charge. DNA and PSS are both strong polyanions with similar linear charge densities. The total number of charges of the two polyanions was kept comparable so that the maximal entropy gain due to counterion release upon complexation would be equal. Single stranded DNA (ssDNA) was not chosen as a flexible analog for dsDNA, in part because they routinely form ill-defined secondary structures,64,65 and in part because of lack of stability. Two contour lengths of polyanion were investigated: one comparable to the micelle radius (∼30 nm) and one much longer (∼1 μm). At these length scales, the DNA are oligomers and linearized plasmids, respectively, both of which are of potential therapeutic interest.66,67 The spherical cationic micelles were preassembled from poly-

(dimethylaminoethyl methacrylate)-block-poly(n-butyl methacrylate) (DMAEMA-b-nBMA).68 The chemical structure of this polymer and PSS are shown in Figure S19 in the Supporting Information. This system was chosen because the core is rubbery, but kinetically frozen with respect to chain exchange. Previous work has shown that cationic micelles with a rubbery core have a higher transfection efficiency compared to those with glassy cores.69 The corona, poly(DMAEMA), is a weak polycation and has proven to be effective in compacting DNA for delivery.70,71 The complexation process was studied by turbidimetric titration. The size and structure of the micelleplexes at different charge ratios and ionic strengths were measured by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM). The ionic strength range from 20 mM to 1 M was covered as a first attempt to map the phase behavior of those complexes. These results provide direct evidence that polyanion flexibility affects the complexation with cationic micelles when the polyanion chain is much longer than the micelle radius, and that DNAmicelleplexes behave similarly to PSS-micelle complexes when the polyanions are short.

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EXPERIMENTAL SECTION Materials. Monomers 2-(dimethylamino)ethyl methacrylate (DMAEMA) and n-butyl methacrylate (BMA), initiators 2,2′azobis(2-methylpropionitrile) (AIBN), 4,4′-azobis(4-cyanovaleric acid) (V-501), and chain transfer agent 4-cyano-4(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid (CDT) were purchased from Sigma-Aldrich, and were either recrystallized or distilled before use. Complementary DNA 20-mers with sequences 5′-AAATCTATCGCTTGTATGGG-3′ and 3′TTTAGATAGCGAACATACCC-5′ were synthesized by Integrated DNA Technologies, Inc. Quick-load Purple 2-Log DNA ladder (0.1−10.0 kb) and HpaI restriction enzyme were purchased from New England BioLabs Inc. A phenol/ chloroform/isoamyl alcohol one-phase mixture (25:24:1 by volume) was purchased from VWR International. Two poly(sodium styrenesulfonate) standards were purchased from PSS Polymer Standards and are denoted as PSS-980 (Mw = 976000 Da, Đ < 1.2) and PSS-30 (Mw = 29500 Da, Đ < 1.2) Polymer Synthesis. The diblock copolymer poly(DMAEMA-b-nBMA) was synthesized by sequential RAFT polymerization as described by Sprouse et al.68 The product was characterized by proton nuclear magnetic resonance spectroscopy (1H NMR) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDITOF-MS), yielding Mn = 41 kg/mol and Đ = 1.09. The Mn of the DMAEMA block is 27 kg/mol and that of the nBMA block is 14 kg/mol. Micelle Formation. Poly(DMAEMA27-b-nBMA14) formed spherical micelles by direct dissolution in deionized water at 1 mg/mL, as described previously.68 Micelle stock solutions were dialyzed against buffers of the desired pH and ionic strength before use. The dialysis membrane had a cutoff of Mw = 6−8 kg/mol and samples were dialyzed against five changes of at least 50-fold volume excess of buffer. The buffering agents were acetic acid, 2-(N-morpholino)ethanesulfonic acid (MES), and 3-(N-morpholino)propanesulfonic acid (MOPS) at 20 mM for buffers at pH 5, 6, and 7, respectively. The final solution ionic strengths were adjusted using sodium chloride. Previous work showed that the micelle cores were kinetically trapped and had a radius of 8 ± 1 nm.68 At pH 7, 100 mM ionic strength, the 6709


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increment was 30 μL. After every 15 additions, 0.45 mL were removed from the titration solution so that desired N/P (or N/ S) ratios could be reached with a reasonable total numbers of additions. The titrations were carried out using a home-built transmission setup that included a HeNe laser, which passed a 632 nm wavelength beam through the titration solution, and a laser power meter (Spex Industries). The solution was stirred during titration using a stir bar at 500 rpm. The titrations were carried out at 1 addition/min and the homogeneity of the solution was inspected visually after each addition. The transmitted laser power was recorded between two additions. To ensure an accurate reading of the laser intensity, stirring was briefly stopped ( 1, the system is net positive, and at N/P (N/S) < 1, the system is net negative. Dynamic Light Scattering. Dynamic light scattering (DLS) was carried out using a Brookhaven Instruments BI200DM multiangle light-scattering instrument with a 637 nm laser. Glass vials for DLS measurements were gently washed using pipe cleaners, triple-rinsed with water and acetone, and then oven-dried before use to reduce the contributions of dust. During measurements, samples were kept at 23 °C. Micelle and polyanion solutions were filtered using 0.2 and 0.45 μm filters, respectively, before measurement. Micelleplexes were prepared using the same method as described in turbidimetric titration, and titrations were stopped at the targeted N/P ratio. To minimize the contribution of dust, the vials in which the micelleplexes were prepared were subjected to the same cleaning steps as described for glass vials used for DLS. The micelle and polyanion solutions used for forming the micelleplexes were prefiltered before the titration. No filtration was conducted after complexation to preserve the structures of the complexes. As Figure S17 shows (Supporting Information), any introduction of dust during titration was minimal and does not affect the interpretation of DLS results for micelleplexes. For micelle and micelleplex samples, correlation functions were acquired at five scattering angles from 60−120° at 15° increments, and were accumulated for at least 10 min per angle. The distributions of hydrodynamic radii were calculated using the Stokes−Einstein relationship from the decay constant distributions, which were obtained by applying the REPES algorithm to intensity correlation functions acquired at 90°.76 The viscosities and refractive indices of the buffers were approximated using sodium chloride solutions of the same ionic strength and temperature. Based on the distributions of the hydrodynamic radii, the correlation function at each angle was fitted with either a second cumulant or a double exponential model, and the obtained mutual diffusion coefficients were

micelles had a hydrodynamic radius of 33 ± 1 nm by dynamic light scattering. By static light scattering, the molar mass of the micelles was measured as (3.8 ± 0.4) × 106 Da, which is equivalent to a maximum of (16 ± 2) × 103 mol amine groups per micelle. In this solution, the second viral coefficient of the micelle was measured to be −(6 ± 2) × 10−5 mol cm3/g2, which reflects relatively poor solvent quality; it is also broadly consistent with previous studies.72−75 The mutual diffusion coefficient of the micelles was found to be independent of the concentration over the range studied below, justifying the interpretation in terms of hydrodynamic radius.68 These micelles were also stable in solution and showed no signs of aggregation over time.68 DNA Oligomer. Double stranded DNA oligomer (hence referred to as L20) was obtained by annealing complementary single-stranded 20-mers. The single-stranded 20-mers were first dissolved in water to make up a 1.6 mM solution. Equal volumes of two 20-mer solutions were then mixed, heated to 90 °C for 2 min and gradually cooled to allow association of complementary strands. The stock solution of the resultant double stranded oligomers was divided into small quantities and lyophilized. Lyophilized oligomers were dissolved in the desired buffer and the concentration of the solution was measured by an ultraviolet−visible light spectrophotometer, given that the oligomer has an absorbance of 0.021 μg cm/mL at 260 nm. Linear DNA. Plasmid DNA with 2442 base pairs (bp) was prepared by amplification in Escherichia coli in TB culture, and then isolated using the PerfectPrep Endofree Plasmid Maxi Kit (VWR, Pennsylvania). Linear double stranded DNA (hence referred to as L2442) was obtained by digesting plasmid with HpaI restriction enzyme using 1 enzyme unit per μg DNA. Digestion took place at 37 °C for 3 h. Complete digestion was confirmed using gel electrophoresis. Undigested and digested plasmids were run in parallel with Purple 2-Log DNA ladder through a 1% agarose gel, as shown in Supporting Information, Figure S1. After electrophoresis, the lane loaded with digested DNA showed one band with the correct size in comparison with the ladder. The lane loaded with undigested plasmid shows three bands, which were absent in the lane loaded with linearized DNA. After digestion, the protein content was removed by phenol extraction. Briefly, the digestion mixture was first mixed with an equal volume of phenol/chloroform/ isoamyl alcohol 25:24:1 mixture, vortexed, and then centrifuged. The top aqueous phase was retained. The remaining organic phase was then mixed with an equal volume of 10 mM Tris buffer (pH 8, 1 mM EDTA), vortexed, and then centrifuged. The top aqueous phase was extracted and the two aqueous phases were combined and re-extracted with an equal volume of fresh phenol/chloroform/isoamyl alcohol mixture. The final aqueous phase containing the linear DNA was isolated from the mixture after vortexing and centrifugation. Before use, linear DNA was dialyzed against deionized water and lyophilized. Dried DNA was dissolved in the desired buffer and its concentration was determined as for the DNA oligomers. Turbidimetric Titration and Micelleplex Formation. Turbidimetric titrations were carried out manually by gradually adding one polyelectrolyte solution to the other. Both solutions were prepared at 0.2 mg/mL and were of the same pH and ionic strength. This concentration was selected to be comparable with previous reports.22,25,27,35,36 In a typical titration, the starting volume was 1.5 mL and the addition 6710


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The Journal of Physical Chemistry B averaged over five angles before converting into the averaged hydrodynamic radii. For DNA solutions, additional correlation functions were acquired at lower angles. The center-of-mass diffusion coefficient of L2442 was obtained by plotting the obtained diffusion coefficient as a function of squared wavevector q2, and using the diffusion coefficient value in the q2 = 0 limit. Cryogenic Transmission Electron Microscopy. An FEI Vitrobot Mark III vitrification system was used to prepare vitrified samples for imaging. First, lacy carbon/Formvar grids (Ted Pella, 300 mesh) were cleaned and hydrophilized using a PELCO easiGlow glow discharge cleaning system. Then the grids were transferred to the climate-controlled chamber in the vitrification system. The climate chamber was kept at 26 °C and 99% humidity. About 3 μL of solution containing complexes was added onto the grid. The loaded grid was then subsequently blotted for 5 s, annealed for 1 s, and plunged into liquid ethane, which was kept at its boiling point and surrounded by liquid nitrogen. The vitrified samples were then transferred and stored in liquid nitrogen, until they were imaged using a FEI Tecnai G2 Spirit BioTWIN microscope coupled with a single-tilt cryo holder. The microscope was operated at 120 kV and images were taken at underfocus for adequate contrast.

Poly(styrenesulfonate) (PSS) was included as a model flexible polyanion to compare with the complexation behavior of DNA. PSS-30 serves as an analog to L20 as it is also a relatively short polyanion. Furthermore, its complexation with related poly(DMAEMA-b-styrene) micelles was thoroughly studied by Laaser et al.25 To a first approximation the poly(DMAEMA-b-nBMA) micelles may be considered equivalent to poly(DMAEMA-b-S) micelles, because it was established that poly(DMAEMA-b-nBMA) micelle cores are kinetically trapped and the corona behavior is independent of the core dynamics.68 Therefore, the complexation of L20 with poly(DMAEMA-b-nBMA) micelles can be closely compared with the study by Laaser et al. with PSS-30.25 In the long chain regime, PSS-980 serves as an analog to L2442 because they have similar total charges, comparable contour lengths, and strong anionic groups, as detailed in Supporting Information (Table S1). The most significant difference is that the PSS chains are more flexible than the double stranded DNA. The intrinsic persistence length of DNA is 50 nm while that of PSS is only 1.4 nm.55,82 The difference in chain flexibility is reflected in the hydrodynamic radii; at 500 mM ionic strength, the Rh of L2442 is 59 nm and that of PSS-980 is 28 nm. This difference in hydrodynamic radii likely exerts a modest influence on the size of the resultant micelleplexes, as both polyanions should undergo significant conformational changes upon complexation, in order to localize around the micelle corona. Complexation with DNA Oligomer L20. The complexation between cationic micelles and DNA was first examined by turbidimetric titration as a function of N/P ratio and addition sequence. As an example, the solution transmittance during forward and reverse titrations between micelles and L20 at pH 5, 20 mM is shown in Figure 1. At varying ionic strengths and

Table 1. Properties of Polyanionsa abbreviation

polyanion

charged units/chain

polyanion to micelle number ratio at 1:1 charge ratio

L20 PSS-30 L2442 PSS-980

linear DNA PSS linear DNA PSS

40 150 4884 4700

375 103 3.1 3.1

a

Table S1 in the Supporting Information includes additional comparisons between PSS-980 and L2442, including the hydrodynamic radii of the polyanions and their linear charge density.

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RESULTS AND DISCUSSION Polyanions. The DNA and PSS polyanions used in this study are described in Table 1. Based on their lengths, the four polyanions can be categorized into two regimes: the short chain regime, in which the polyanion contour length is shorter than or comparable to the corona length of the micelles, and the long chain regime, where the polyanion contour length is much longer than the radius of the micelle. The poly(DMAEMA-bnBMA) micelle has a core radius of 8 nm and corona thickness of around 20 nm, depending on the solution ionic strength.68 The B-form double-stranded DNA has two negative charges and a rise of 0.34 nm per base pair. In solutions of 20 mM to 1 M ionic strength, the persistence length of DNA is about 50 nm,77,78 and thus the DNA oligomer L20 is rod-like with a contour length of about 7 nm, while the linear DNA L2442 has a contour length of about 830 nm, which is an order-ofmagnitude larger than the micelle radius. L2442 should adopt a coil conformation in solution. It is established that the centerof-mass diffusion coefficient of DNA is essentially independent of solution ionic strength in the range of 20−500 mM.79−81 Within this range, DLS of L2442 yielded a center-of-mass diffusion coefficient of 3.8 × 10−12 m2/s, which agrees well with a previous report for linear DNA of similar length.80 More details are provided in Supporting Information. At 100 mM, this corresponds to a hydrodynamic radius of 61 nm.

Figure 1. Turbidimetric titration curves of poly(DMAEMA-b-nBMA) micelles with L20 DNA oligomers in pH 5 acetic acid buffer at 20 mM ionic strength. The dashed line at transmittance of 0.9 shows the titration end points. Vertical error bars are uncertainties in the readings of the transmitted light intensity and horizontal error bars are calculated as the uncertainties in N/P ratio due to repeated pipetting.

pH values, the titration curves between the micelles and L20 show common features, as documented in Supporting Information Figures S3−4. From either direction, the solution was homogeneous until near the charge neutral point (N/P ∼ 1), where precipitates formed. Upon addition of further oppositely charged polyelectrolyte beyond the charge neutral point, the precipitates did not dissolve. The forward and reverse titration curves do not overlap at the same range of N/P ratio, 6711


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N/P = 1, as shown in Figure 2a, but the inhomogeneous region widened asymmetrically with respect to stoichiometric ratio; the inhomogeneous region widened more significantly at N/P < 1 than at N/P > 1. With increasing solution pH, the titration centers remain near N/P ≈ 1, as shown in Figure S19 in the Supporting Information, even though the apparent pKa of the amine groups predicts that at N/P = 1 the +/− charge ratios are less than 1.89 This suggests that the pKa of the amine groups increases in the presence of L20, consistent with previous literature.90−93 In the soluble regions of the phase diagram, the size and structure of the micelleplexes depend on the ionic strength. Figure 3 shows size distributions and cryo-TEM images of L20 micelleplexes at N/P > 1 (net positive). At low ionic strength (20 mM), micelleplexes show bimodal size distributions (Figure 3a), with one population smaller and one population larger than the bare micelle. The corona could contract significantly upon complexation, which contributes to the smaller size, as observed previously.25,94 In general, any free polyanions in the solution will contribute much less to the scattering signal compared to micelles and micelleplexes, and therefore are not revealed in DLS analysis. Cryo-TEM also shows two populations corresponding to single-micelle and multiple-micelle complexes, as shown in Figure 3b. Since the scattering intensity measured by DLS is roughly proportional to the molar mass times the mass concentration of the particle,95 the weight fraction of single-micelle complexes can be estimated from the intensity ratio of the two peaks to be about 75% (more details are given in Supporting Information). Both techniques indicate that the majority of the complexes are single-micelle complexes. At higher ionic strength (500 mM, Figure 3c), micelleplexes were monodisperse from day 1 with size distributions similar to that of single micelles. Cryo-TEM images also showed single-micelle-like particles. At this high ionic strength, titrations between micelles and L20 showed precipitate formation around N/P ∼ 1 as a result of complexation, as L20 and micelles were stable separately under these conditions. Therefore, micelleplexes formed at 500 mM were single-micelle complexes at N/P ∼ 4 as suggested by DLS and cryo-TEM. At both ionic strengths, the micelleplexes were stable and soluble over at least one month.

which suggests that the complexation process depends on the addition sequence and is under kinetic control on the time scale of the titration. The observed solubility of the micelleplexes as a function of charge ratio agrees well with previous studies on linear interpolyelectrolyte complexes,13,14,83,84 and complexes between linear and micellar polyelectrolytes.22,33,85 It is also well established that such complexes are typically kinetically limited at low ionic strength.23,34,86−88 To compare titration curves between different polyelectrolyte pairs and at different pH and ionic strengths, a titration end point is defined as the N/P ratio at which the solution transmittance first falls below 0.9. Precipitates were observed soon after the titration end points. The average of the two titration end points is defined as the titration center. Solution ionic strength and pH affect the solubility of micelleplexes, as outlined by the titration end points versus N/ P ratio; Figure 2 shows the end points of poly(DMAEMA-b-

Figure 2. Titration end points of L20 oligomers with poly(DMAEMAb-nBMA) micelles as a function of ionic strength at pH 5. The shaded area is a schematic illustration of the inhomogeneous regions in the titrations.

nBMA) micelles and L20 as a function of ionic strength. With increasing ionic strength, the titration centers remained around

Figure 3. (a),(c) Size distributions and (b),(d) cryo-TEM images of micelleplexes formed between poly(DMAEMA-b-nBMA) micelles and L20 DNA oligomers at N/P ∼ 4 in pH 5 buffers at (a),(b) 20 mM ionic strength and (c),(d) 500 mM ionic strength. The scale bars are 200 nm. 6712


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Figure 4. Temporal evolution of L20 micelleplexes at 20 mM ionic strength. The micelleplexes were formed at (a) N/P = 0.6, 0.6, and 0.5 and (b) N/P = 3.0, 2.7, and 2.5 in pH = 5, 6, and 7 buffers, respectively. The dotted lines show the Rh of micelles in corresponding buffers and the “x” indicates the appearance of precipitates.

Figure 5. Schematic illustration of the structural evolution of micelleplexes formed with DNA oligomers.

advantage, and reduces the overall translational entropy of the L20. Over time, L20 can rearrange around the micelles and the excess L20 would be free to return to the solution. Without overcharging, complexes are more likely to aggregate and precipitate. Experimentally, the micelleplexes at N/P < 1 increased in size and eventually precipitated, consistent with the scenario proposed in Figure 5. At N/P > 1, micelles are in excess and the kinetic products could include single-micelle complexes and multiple-micelle complexes bridged by L20. In addition, there might be uncomplexed micelles coexisting with micelleplexes. Experimentally, in 20 mM ionic strength buffers, micelleplexes at N/ P > 1 showed bimodal size distributions with one population corresponding to single micelles and one corresponding to multiple-micelle complexes. Over time, L20 can redistribute equally among all the micelles. The resulting thermodynamic products are single-micelle complexes that are solubilized by the excess charge of the micelle corona. Such structures were observed at high ionic strength and N/P > 1, where only singlemicelle complexes were formed. This temporal evolution of the micelleplexes is very similar to that of complexes formed between PSS-30 and poly(DMAEMA-b-PS), as recently reported by Laaser et al.25 Those complexes also exhibited long-term stability (over three months) with micelles in excess, and tended to aggregate with PSS-30 in excess. This similarity between the two systems indicates that in the short chain

While the structure of the micelleplexes depends on the ionic strength, their long-term stability is dictated by the N/P ratio. At 20 mM, micelleplexes were initially soluble, as shown in Figure 4, but showed markedly different temporal evolution depending on N/P ratio. At N/P < 1 (net negative), L20 is in excess. The micelleplexes initially exhibited monomodal size distributions as shown in Figure 4a. Over time, the size of the complexes increased and the distribution became bimodal. Precipitation was observed as early as day 14. Once the solutions precipitated, DLS was measured on the upper clear solutions. Eventually after 28 days, the scattering intensity of the upper solution became too weak for DLS measurements and solid white precipitates were found at the bottom. In comparison, micelleplexes at N/P > 1 showed stable bimodal size distributions over a month, as shown in Figure 4b. With micelles or L20 in excess, the solution pH did not affect the size distribution or the long-term stability of the micelleplexes. Comparison to PSS-30 Micelleplexes. The temporal evolution can be rationalized as a process in which micelleplexes formed under kinetic control evolve toward the thermodynamic structures, as illustrated in Figure 5. At low ionic strength such as 20 mM, the complexation process is likely to be kinetically limited. At N/P < 1, L20 is in excess and the kinetic products could include overcharged micelles and bridged micelles. Since the entropy gain of counterion release is the main driving force for complexation, overcharging beyond the charge-neutral point provides little to no energetic 6713


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1 and the end points for N/P < 1 fell much below N/P = 1. In comparison, the titration centers of flexible PSS-980 are symmetric around N/P = 1 and the middle inhomogeneous region widens symmetrically around N/P = 1 with increasing ionic strength. Therefore, compared to PSS-980, the titration end points of L2442 were shifted toward lower N/P ratios for both titration directions. Since PSS-980 and L2442 have similar total numbers of charges and contour lengths but differ significantly in their intrinsic persistence lengths, the downward shifting of the titration centers of L2442 is attributable to chain stiffness. The micelleplexes of L2442 and PSS-980 not only exhibited different solubility envelopes, but also differed in their structures, as shown in Figure 7. At 500 mM ionic strength, where the solubility envelopes differed the most, the complexes of both polyanions showed bimodal size distributions when micelles are in excess, but the complexes formed with PSS-980 are significantly bigger than those formed with L2442. CryoTEM images of the two complexes further illustrate structural differences. As Figure 7b shows, the complexes formed with flexible PSS-980 had a large number of micelles closely associated with neighboring micelles. In comparison, the micelleplexes formed with stiff L2442, as shown in Figure 7c, were mostly single-micelle-like particles (more cryo-TEM images are in Figure S11). The rare multiple-micelle complexes only comprised a few micelles, and contributed to the population of larger complexes as shown in Figure 7. Due to the low contrast, DNA molecules were not always clearly visualized in cryo-TEM images. However, micelles in close vicinity were likely bridged by DNA molecules, because otherwise they should be randomly dispersed, as shown by Sprouse et al. 68 Representative cryo-TEM images of micelleplexes are also shown in Figure S11 in Supporting Information with multiple-micelle micelleplexes highlighted. Both complexes of L2442 and PSS-980 were stable, and the structure difference persisted over a month, as shown in Figure S14. This structural difference between micelleplexes of L2442 and PSS-980 is apparently caused by the flexibility difference between the polyanions. This interpretation rests on the observations that the double helix structure of dsDNA was not globally heavily distorted or disassembled when complexed with the cationic micelles, as indicated by the similarity in the circular dichroism spectrum of micelleplexes compared to dsDNA, illustrated in Figure S20 in the Supporting Information. The initial size difference between the two polyanions cannot be the primary reason for this structural difference, because the complexes of PSS-980 were bigger than micelleplexes of L2442 but the hydrodynamic radius of PSS980 at 500 mM is smaller than that of L2442. At these ionic strengths and N/P ratios, the complexation of L2442 to micelles is apparently complete, with no free L2442 in the solution as indicated by electrophoresis (see Figure S13). At low ionic strength, the structural differences between the complexes formed with L2442 and PSS-980 persist. In addition, the stability of the complexes differs between polyanions. Figure 8a and c shows the size distributions of micelleplexes formed with L2442, with either micelles or L2442 in excess, respectively. In the former case, the micelleplexes were monodisperse with an average hydrodynamic radius larger than single micelles. Cryo-TEM of these micelleplexes, shown in Figure 9a, also shows single-micelle like particles and small multiple-micelle complexes (more cryo-TEM images are in Figure S12). The single population measured by DLS therefore

regime the stiffness of the polyanion chain does not affect the structure, or the stability, of the complexes. Solubility of L20 micelleplexes as a function of N/P ratio, by turbidimetric titration, quantitatively agrees with previous literature but shows quantitative differences from that of PSS30 complexes. The titration centers for the micelleplexes were found to be around N/P = 1, but the inhomogeneous region widened with increasing ionic strength. This phase behavior qualitatively agrees with previous literature on interpolyelectrolyte complexation.16,96,13,14,83,84 Previous work on the complexation of PSS-30 with poly(DMAEMA-b-S) micelles also showed similar trends.25 However, there is one quantitative difference between the “titration phase diagrams” of L20 micelleplexes and PSS-30-micelle complexes. With increasing ionic strength, the inhomogeneous region of PSS-30 complexes widens symmetrically around N/P = 1 while the inhomogeneous region of L20 micelleplexes widens asymmetrically, with the titration end points for N/P < 1 decreasing more significantly with increasing ionic strength. This may be due to the fact that L20 is shorter than PSS-30 and at the same ionic strength, micelleplexes of L20 are closer to thermodynamic equilibrium than those of PSS-30 due to higher association− dissociation rates of shorter polyanion chains. As proposed earlier, micelleplexes at thermodynamic equilibrium solubilize as single-micelle complexes at N/P > 1. At 500 mM, micelleplexes of L20 formed uniform single-micelle complexes on the same day the complexes were made, but complexes of PSS-30 with poly(DMAEMA-b-S) showed bimodal size distributions on day 1.25 Additionally, asymmetric titration curves were also observed between very short (1 kg/mol) PSS chains and poly(DMAEMA-b-S) micelles.36 Since the complexation of L20 is closer to thermodynamic equilibrium, the titrations reflect the fact that at thermodynamic equilibrium the micelleplexes are insoluble at N/P < 1, and therefore the insoluble regions widened more significantly. Complexation of Long Polyanions. Compared to L20, L2442 is much longer, with a contour length 1 order of magnitude larger than the diameter of the poly(DMAEMA-bnBMA) micelles. The turbidimetric titrations of L2442 with the micelles exhibit different solubility phase diagrams from those of L20, as shown in Figure 6. While the middle inhomogeneous region widens with increasing ionic strength, similar to L20, the titration end points of L2442 for N/P > 1 were close to N/P =

Figure 6. Titration end points versus solution ionic strength at pH 5 for L2442 and PSS-980 with poly(DMAEMA-b-nBMA) micelles. Full titration curves are shown in Figures S9−10. 6714


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Figure 7. (a) Hydrodynamic radii and (b),(c) cryo-TEM images of micelleplexes of (b) PSS-980 and (c) L2442 formed in pH 5 buffers at 500 mM ionic strength with (b) N/S = 5.3 and (c) N/P = 5.3. The scale bars are 200 nm.

Figure 8. Hydrodynamic radii of micelleplexes formed with L2442 (a),(c) and PSS-980 (b),(d) in pH 5, 20 mM ionic strength buffer at (a),(b) N/P or N/S = 5.4 and (c),(d) N/P or N/S = 0.2. Micelleplexes of L2442 at N/P = 0.2 showed precipitation at day 14. DLS was measured using the upper clear solution when precipitation was observed.

were stable in the solution for over a month, as shown in Figure 8d. The lack of evolution of these complexes suggests that they were kinetically trapped upon formation at this low ionic strength. The experimental observations, as a function of variables such as solution ionic strength, N/P ratios, polyanion length, and flexibility, are summarized in Table S4 in the Supporting Information. As the complexation of oppositely charged polyelectrolytes is primarily driven by the entropy gain from counterion release, at high ionic strength this driving force is diminished to the extent that no complexation will occur.97−100 For L2442 and the poly(DMAEMA-b-nBMA) micelles, it was found that 1 M ionic strength was sufficient to arrest complexation. At 1 M ionic strength, turbidimetric titrations between L2442 and the micelles showed that the solution transmittance was constant during the titrations from both directions, as shown in Figure 10a. This indicates that no complexation occurred at any N/P ratio. DLS results further support this conclusion, as shown in Figure 10b. With the addition of L2442 to the micelle solution at 1 M ionic strength, the particle size did not change and was the same as the micelles, while at 500 mM ionic strength

includes uncomplexed micelles, possibly single-micelle complexes and multiple-micelle complexes, which are small in both size and number. These micelleplexes formed with micelles in excess were stable and soluble in solution for at least two months. When L2442 was in excess, the micelleplexes initially showed bimodal size distributions and precipitates were observed by day 14. The cryo-TEM image of those micelleplexes shown in Figure 9c reveals large, tightly structured aggregates that are most likely composed of multiple micelles and L2442. Compared to L2442, micelleplexes formed with flexible PSS980 were stable with either micelles or PSS in excess at 20 mM ionic strength, as shown in Figure 8b and d. With PSS-980 in excess, complexes showed bimodal size distributions similar to those observed at 500 mM, with one population of multiplemicelle complexes and one of single-micelle complexes and uncomplexed micelles. With micelles in excess, the complexes again showed bimodal size distributions. Cryo-TEM images in Figure 9b and d show one population of single-micelle complexes and one population of multiple-micelle complexes. Unlike micelleplexes with L2442 in excess, these complexes 6715


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charge neutrality. However, the solution transmittances were direction-dependent, which suggests that the complexation process is still under kinetic control. At N/S = 5, the DLS of the mixture showed the characteristic bimodal size distributions similar to complexes at lower ionic strengths. Therefore, at 1 M ionic strength complexation between PSS-980 and poly(DMAEMA-b-nBMA) micelles still proceed under kinetic control, but no complexation occurs between L2442 and the same cationic micelles. Effects of Polyanion Flexibility. There are significant differences between complexes of L2442 and PSS-980 in terms of titration curves, structure, and long-term stability. We attribute these primarily to the differing flexibilities. Furthermore, we hypothesize that the stiffness of a polyanion affects its complexation with a cationic micelle in two ways. First, flexibility affects the structure, which in turn affects the composition and aggregation tendency of the complexes. Second, the stiffness of the polyanion incurs an extra energy penalty for complexation, in terms of bending to maximize charge matching, which diminishes the net driving force for complexation. We propose that these two factors together contribute to the shift of the titration centers of L2442 to lower N/P ratios compared to PSS-980, as well as the structural and long-term stability differences between micelleplexes of L2442 and PSS-980. The flexibility of PSS facilitates aggregation of complexes by two mechanisms, as shown schematically in Figure 11. If two

Figure 9. Cryo-TEM images of complexes formed with L2442 (a),(c) and PSS-980 (b),(d) in pH 5, 20 mM ionic strength buffer at (a),(b) N/P or N/S = 5.4 and (c),(d) N/P or N/S = 0.2. Cryo-TEM images (c),(d) were taken at day 1 and scale bars are 200 nm.

Figure 11. Schematic illustration of micelleplex formation with long polyanions at N/P > 1.

micelles were bridged by one PSS chain, the bridged chain could condense into the micelle corona to maximize ion pairing, pulling the two micelles together to form one aggregate. Alternatively, condensed PSS chains can form neutralized patches in the micelle corona, which reduces the electrostatic repulsion between micelles and promotes aggregation. As a result, at N/S > 1 many complexes formed by PSS-980 and cationic micelles are large multiple-micelle complexes that precipitate before reaching N/S = 1. In contrast, dsDNA has a persistence length around 50 nm and cannot easily fully condense in the corona, which inhibits the aggregation of single-micelle complexes, as illustrated in Figure 11. Therefore, micelles bridged by one L2442 chain will remain at a distance. In addition, DNA cannot form neutralized patches on these micelles as its intrinsic stiffness prevents compact folding. Instead, loops and trains of DNA could extend outside the corona, as has been seen in simulations of complexation

Figure 10. (a) Turbidimetric titrations of PSS-980 and L2442 with poly(DMAEMA-b-nBMA) micelles in pH 5 buffer at 1 M ionic strength and (b) the measured hydrodynamic radii of the particles for N/P or N/S = 5. In (a), the solid symbols indicate the solution was homogeneous and the open symbols indicate the solution was inhomogeneous. The circles show solution transmittance when polyanion was added to micelle solution, and the square the reverse.

micelleplexes at the same N/P ratio showed clear bimodal size distributions. In comparison, complexation between PSS-980 and poly(DMAEMA-b-nBMA) micelles still proceeded at 1 M ionic strength, and even at 3 M ionic strength, as shown in Figure S21. Turbidimetric titrations showed that solution transmittance decreased and eventually led to precipitation near 6716



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CONCLUSIONS In complexation of linear polyanions with cationic polymeric micelles, the flexibility of the polyanion chain plays an important role in determining the structure and stability of the complexes in the regime where the polyanion is much longer than the radius of the cationic micelles. The stiffness of the polyanion chain reduces the thermodynamic driving force for complexation and changes the structure of the complexes, as the stiffness increases the difficulty of condensing the polyanion around a micelle. Consequently, micelleplexes show much higher populations of single-micelle complexes when micelles are in excess, and an elevated tendency to aggregate with DNA in excess. While the precipitation regions of PSS complexes are around the charge neutral point (N/S ∼ 1), the precipitation regions of DNA micelleplexes shift toward lower N/P ratios. In the regime where the polyanion chain length is shorter than or comparable to micelle corona length, the properties of the complexes no longer depend on the flexibility of the polyanion chain. Complexes of DNA oligomers and short PSS chains share similar titration curves as a function of solution ionic strength and similar structural evolution to thermodynamically more favored states as a function of charge ratio between the two polyelectrolytes. Overall, these results bring insight into the fundamental principles governing how chain stiffness controls the complexation between dissimilar polyelectrolyte pairs, draw direct comparisons between flexible polyanion PSS and dsDNA in the context of complexing with cationic polymeric micelles, and should help guide future design of polymer−colloid interpolyelectrolyte complexes, such as those that might be used for gene delivery applications.

between stiff polyelectrolytes and oppositely charged spheres.57,59,101 As a consequence, the micelleplexes of L2442 with micelles in excess were predominantly small complexes (most likely single-micelle complexes) and the precipitation of L2442 micelleplexes was delayed to close to the charge neutral point. Besides micelleplex structure, flexibility also affects the thermodynamic driving force for complexation, which can be assessed using the critical ionic strength for complexation.99−101 The lower critical ionic strength for a DNA-micelle pair suggests a weaker driving force for complexation, which we hypothesize reflects an extra energetic penalty for stiff chains to complex with a micelle, as DNA needs to bend to accommodate more charge pairs in the micelle corona. An order-of-magnitude estimation of energy cost for DNA bending is included in the Supporting Information. This extra energetic penalty potentially also contributes to poor colloidal stability of micelleplexes at N/P < 1. At the same ionic strength, L2442 micelleplexes precipitated earlier than PSS complexes and therefore reached the thermodynamically favored state more rapidly, as shown in Figure 5. Since the evolution depends on the rate of disassociation and reassociation of the polyanion, the faster evolution for DNA implies a lower activation energy for a DNA chain to totally dissociate from a micelle, because DNA chains recover the extra bending energy. The significant impact of chain flexibility observed here is unique to linear-micellar systems, due to the high geometrical and charge density disparities between two polyelectrolytes. For linear−linear systems, chain flexibility has been found to have a much more limited influence,83,102 and did not affect the centering of the precipitation regions around charge neutrality.83,103 The influence of chain flexibility on the complexation between dissimilar polyelectrolytes was also captured experimentally by Störkle et al. in a study of complexation between DNA and cationic cylindrical brushes.20 They observed that the DNA-brush complexes with excess DNA were significantly less overcharged compared to DNA-linear polycation complexes, at low ionic strength. For linear-surfactant micelle systems, stiffer chains were also found to have a weaker binding strength.56 The influence of polyanion flexibility on complexation with cationic micelles was only evident for long polyanions. For short chains the stiffness of the polyanion does not affect the properties of the complexes, which is reasonable as the short chain does not need to undergo conformational changes. Comparing short and long chains, the titration curves of PSS-30 are similar to those of PSS-980, as shown in Figure S15, whereas those of L2442 deviate from the titration curves of L20, as shown in Figure S16. With increasing DNA length, the energy cost for bending DNA for complexation increases but the entropy gain from counterion release also increases. Although both L20 and L2442 complex with the cationic micelles at 500 mM, the inhomogeneous region of L20 is wider than that of L2442. For flexible chains, the chain length matters less as both chain lengths are significantly longer than their persistence length. The persistence length of PSS is comparable to those of single strand DNA (ssDNA) and RNA,104 and therefore its complexation behavior may represent those of ssDNA and RNA, rather than dsDNA, in the context of complexation with cationic micelles. Investigation of the influence of DNA length in the long chain regime, as well as a comparison between linear and supercoiled DNA, in the complexation with cationic micelles is currently under way.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b03732. Characterization of linear DNA, turbidimetric titrations, cryo-TEM images, and analysis of dynamic light scattering data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jennifer E. Laaser: 0000-0002-0551-9659 Theresa M. Reineke: 0000-0001-7020-3450 Timothy P. Lodge: 0000-0001-5916-8834 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported primarily by the National Science Foundation through the University of Minnesota MRSEC under Award Number DMR-1420013. Part of this work was carried out in the College of Science and Engineering Characterization Facility, University of Minnesota, which has received capital equipment funding from NSF through the UMN MRSEC program under Award Number DMR-1420013. J.E.L. was supported in part by the L’Oréal for Women in Science postdoctoral fellowship program, and Y. J. received a Louise T. Dosdall Fellowship. 6717


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(21) Xu, Y.; Borisov, O. V.; Ballauff, M.; Müller, A. H. E. Manipulating the Morphologies of Cylindrical Polyelectrolyte Brushes by Forming Interpolyelectrolyte Complexes with Oppositely Charged Linear Polyelectrolytes: An AFM Study. Langmuir 2010, 26, 6919− 6926. (22) Chelushkin, P. S.; Lysenko, E. A.; Bronich, T. K.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Polyion Complex Nanomaterials from Block Polyelectrolyte Micelles and Linear Polyelectrolytes of Opposite Charge: 1. Solution Behavior. J. Phys. Chem. B 2007, 111, 8419−8425. (23) Chelushkin, P. S.; Lysenko, E. A.; Bronich, T. K.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Polyion Complex Nanomaterials from Block Polyelectrolyte Micelles and Linear Polyelectrolytes of Opposite Charge. 2. Dynamic Properties. J. Phys. Chem. B 2008, 112, 7732− 7738. (24) Zhiryakova, M. V.; Izumrudov, V. A. Water-Soluble Polyelectrolyte Complexes of Astramol Poly(propyleneimine) Dendrimers with Poly(methacrylate) Anion. J. Phys. Chem. B 2014, 118, 13760− 13769. (25) Laaser, J. E.; Jiang, Y.; Petersen, S. R.; Reineke, T. M.; Lodge, T. P. Interpolyelectrolyte Complexes of Polycationic Micelles and Linear Polyanions: Structural Stability and Temporal Evolution. J. Phys. Chem. B 2015, 119, 15919−15928. (26) Pergushov, D. V.; Müller, A. H. E.; Schacher, F. H. Micellar Interpolyelectrolyte Complexes. Chem. Soc. Rev. 2012, 41, 6888. (27) Burkhardt, M.; Ruppel, M.; Tea, S.; Drechsler, M.; Schweins, R.; Pergushov, D. V.; Gradzielski, M.; Zezin, A. B.; Müller, A. H. E. WaterSoluble Interpolyelectrolyte Complexes of Polyisobutylene-blockPoly(methacrylic acid) Micelles: Formation and Properties. Langmuir 2008, 24, 1769−1777. (28) van der Maarel, J. R. C.; Groenewegen, W.; Egelhaaf, S. U.; Lapp, A. Salt-Induced Contraction of Polyelectrolyte Diblock Copolymer Micelles. Langmuir 2000, 16, 7510−7519. (29) Förster, S.; Hermsdorf, N.; Böttcher, C.; Lindner, P. Structure of Polyelectrolyte Block Copolymer Micelles. Macromolecules 2002, 35, 4096−4105. (30) Förster, S.; Abetz, V.; Müller, A. H. E. Polyelectrolyte Block Copolymer Micelles. In Polyelectrolytes with Defined Molecular Architecture II; Springer: Berlin Heidelberg, 2004; Vol. 166, pp 173− 210, DOI: 10.1007/b11351. (31) Zhang, L.; Barlow, R. J.; Eisenberg, A. Scaling Relations and Coronal Dimensions in Aqueous Block Polyelectrolyte Micelles. Macromolecules 1995, 28, 6055−6066. (32) Moffitt, M.; Khougaz, K.; Eisenberg, A. Micellization of Ionic Block Copolymers. Acc. Chem. Res. 1996, 29, 95−102. (33) Lysenko, E. A.; Chelushkin, P. S.; Bronich, T. K.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Formation of Multilayer Polyelectrolyte Complexes by Using Block Ionomer Micelles as Nucleating Particles. J. Phys. Chem. B 2004, 108, 12352−12359. (34) Talingting, M. R.; Voigt, U.; Munk, P.; Webber, S. E. Observation of Massive Overcompensation in the Complexation of Sodium Poly(styrenesulfonate) with Cationic Polymer Micelles. Macromolecules 2000, 33, 9612−9619. (35) Sharma, R.; Lee, J. S.; Bettencourt, R. C.; Xiao, C.; Konieczny, S. F.; Won, Y. Y. Effects of the Incorporation of a Hydrophobic Middle Block into a PEG−Polycation Diblock Copolymer on the Physicochemical and Cell Interaction Properties of the Polymer− DNA Complexes. Biomacromolecules 2008, 9, 3294−3307. (36) Laaser, J. E.; Lohmann, E.; Jiang, Y.; Reineke, T. M.; Lodge, T. P. Architecture-Dependent Stabilization of Polyelectrolyte Complexes between Polyanions and Cationic Triblock Terpolymer Micelles. Macromolecules 2016, 49, 6644. (37) Weaver, J. V. M; Tang, Y.; Liu, S.; Iddon, P. D.; Grigg, R.; Billingham, N. C.; Armes, S. P.; Hunter, R.; Rannard, S. P. Preparation of Shell Cross-Linked Micelles by Polyelectrolyte Complexation. Angew. Chem., Int. Ed. 2004, 43, 1389−1392. (38) Lutz, J. F.; Geffroy, S.; von Berlepsch, H.; Böttcher, C.; Garnier, S.; Laschewsky, A. Investigation of a Dual Set of Driving Forces (Hydrophobic + Electrostatic) for the Two-Step Fabrication of Defined Block Copolymer Micelles. Soft Matter 2007, 3, 694−698.

REFERENCES

(1) Fu, J.; Schlenoff, J. B. Driving Forces for Oppositely Charged Polyion Association in Aqueous Solutions: Enthalpic, Entropic, but Not Electrostatic. J. Am. Chem. Soc. 2016, 138, 980−990. (2) Bucur, C. B.; Sui, Z.; Schlenoff, J. B. Ideal Mixing in Polyelectrolyte Complexes and Multilayers: Entropy Driven Assembly. J. Am. Chem. Soc. 2006, 128, 13690−13691. (3) Böhm, N.; Kulicke, W. M. Optimization of the Use of Polyelectrolytes for Dewatering Industrial Sludges of Various Origins. Colloid Polym. Sci. 1997, 275, 73−81. (4) Bolto, B.; Gregory, J. Organic Polyelectrolytes in Water Treatment. Water Res. 2007, 41, 2301−2324. (5) Antipov, A. A.; Sukhorukov, G. B. Polyelectrolyte Multilayer Capsules as Vehicles with Tunable Permeability. Adv. Colloid Interface Sci. 2004, 111, 49−61. (6) Peyratout, C. S.; Dähne, L. Tailor-Made Polyelectrolyte Microcapsules: From Multilayers to Smart Containers. Angew. Chem., Int. Ed. 2004, 43, 3762−3783. (7) Zhao, Q.; An, Q. F.; Ji, Y.; Qian, J.; Gao, C. Polyelectrolyte Complex Membranes for Pervaporation, Nanofiltration and Fuel Cell Applications. J. Membr. Sci. 2011, 379, 19−45. (8) Ahmadiannamini, P.; Li, X.; Goyens, W.; Joseph, N.; Meesschaert, B.; Vankelecom, I. F. J. Multilayered Polyelectrolyte Complex Based Solvent Resistant Nanofiltration Membranes Prepared from Weak Polyacids. J. Membr. Sci. 2012, 394−395, 98−106. (9) Schaaf, P.; Schlenoff, J. B. Saloplastics: Processing Compact Polyelectrolyte Complexes. Adv. Mater. 2015, 27, 2420−2432. (10) Luo, F.; Sun, T. L.; Nakajima, T.; Kurokawa, T.; Zhao, Y.; Sato, K.; Ihsan, A. B.; Li, X.; Guo, H.; Gong, J. P. Oppositely Charged Polyelectrolytes Form Tough, Self-Healing, and Rebuildable Hydrogels. Adv. Mater. 2015, 27, 2722−2727. (11) Delcea, M.; Möhwald, H.; Skirtach, A. G. Stimuli-Responsive LbL Capsules and Nanoshells for Drug Delivery. Adv. Drug Delivery Rev. 2011, 63, 730−747. (12) Kim, S. H.; Jeong, J. H.; Lee, S. H.; Kim, S. W.; Park, T. G. Local and Systemic Delivery of VEGF siRNA Using Polyelectrolyte Complex Micelles for Effective Treatment of Cancer. J. Controlled Release 2008, 129, 107−116. (13) Zhang, Y.; Yildirim, E.; Antila, H. S.; Valenzuela, L. D.; Sammalkorpi, M.; Lutkenhaus, J. L. The Influence of Ionic Strength and Mixing Ratio on the Colloidal Stability of PDAC/PSS Polyelectrolyte Complexes. Soft Matter 2015, 11, 7392−7401. (14) Dautzenberg, H. Polyelectrolyte Complex Formation in Highly Aggregating Systems. 1. Effect of Salt: Polyelectrolyte Complex Formation in the Presence of NaCl. Macromolecules 1997, 30, 7810− 7815. (15) Spruijt, E.; Leermakers, F. A. M.; Fokkink, R.; Schweins, R.; van Well, A. A.; Cohen Stuart, M. A.; van der Gucht, J. Structure and Dynamics of Polyelectrolyte Complex Coacervates Studied by Scattering of Neutrons, X-rays, and Light. Macromolecules 2013, 46, 4596−4605. (16) Zhang, R.; Shklovskii, B. I. Phase Diagram of Solution of Oppositely Charged Polyelectrolytes. Phys. A 2005, 352, 216−238. (17) Thünemann, A. F.; Müller, M.; Dautzenberg, H.; Joanny, J.; Löwen, H. Polyelectrolyte Complexes. In Polyelectrolytes with Defined Molecular Architecture II; Springer: Berlin Heidelberg, 2004; Vol. 166, pp 113−171, DOI: 10.1007/b11350. (18) Pergushov, D. V.; Borisov, O. V.; Zezin, A. B.; Müller, A. H. E. Interpolyelectrolyte Complexes Based on Polyionic Species of Branched Topology. In Adv. Polym. Sci.; 2010; pp 241131−161, DOI: 10.1007/12_2010_102. (19) Pergushov, D. V.; Remizova, E. V.; Feldthusen, J.; Zezin, A. B.; Müller, A. H. E.; Kabanov, V. A. Novel Water-Soluble Micellar Interpolyelectrolyte Complexes. J. Phys. Chem. B 2003, 107, 8093− 8096. (20) Störkle, D.; Duschner, S.; Heimann, N.; Maskos, M.; Schmidt, M. Complex Formation of DNA with Oppositely Charged Polyelectrolytes of Different Chain Topology: Cylindrical Brushes and Dendrimers. Macromolecules 2007, 40, 7998−8006. 6718


Article

The Journal of Physical Chemistry B (39) Schaffert, D.; Wagner, E. Gene Therapy Progress and Prospects: Synthetic Polymer-Based Systems. Gene Ther. 2008, 15, 1131−1138. (40) Park, T.; Jeong, J.; Kim, S. Current Status of Polymeric Gene Delivery Systems☆. Adv. Drug Delivery Rev. 2006, 58, 467−486. (41) Burke, P. A.; Pun, S. H.; Reineke, T. M. Advancing Polymeric Delivery Systems Amidst a Nucleic Acid Therapy Renaissance. ACS Macro Lett. 2013, 2, 928−934. (42) Bertin, A. Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors. Adv. Polym. Sci. 2013, 256, 103−195. (43) Ibraheem, D.; Elaissari, A.; Fessi, H. Gene Therapy and DNA Delivery Systems. Int. J. Pharm. 2014, 459, 70−83. (44) Desnick, R. J.; Schuchman, E. H. Gene Therapy for Genetic Diseases. Pediatr. Int. 1998, 40, 191−203. (45) Nelson, C. E.; Hakim, C. H.; Ousterout, D. G.; Thakore, P. I.; Moreb, E. A.; Rivera, R. M. C.; Madhavan, S.; Pan, X.; Ran, F. A.; Yan, W. X.; et al. In Vivo Genome Editing Improves Muscle Function in a Mouse Model of Duchenne Muscular Dystrophy. Science 2016, 351, 403−407. (46) Kabanov, A. V. Taking Polycation Gene Delivery Systems from In Vitro to In Vivo. Pharm. Sci. Technol. Today 1999, 2, 365−372. (47) Kakizawa, Y.; Kataoka, K. Block Copolymer Micelles for Delivery of Gene and Related Compounds. Adv. Drug Delivery Rev. 2002, 54, 203−222. (48) Alhoranta, A. M.; Lehtinen, J. K.; Urtti, A. O.; Butcher, S. J.; Aseyev, V. O.; Tenhu, H. J. Cationic Amphiphilic Star and Linear Block Copolymers: Synthesis, Self-Assembly, and in Vitro Gene Transfection. Biomacromolecules 2011, 12, 3213−3222. (49) Mao, C. Q.; Du, J. Z.; Sun, T. M.; Yao, Y. D.; Zhang, P. Z.; Song, E. W.; Wang, J. A Biodegradable Amphiphilic and Cationic Triblock Copolymer for the Delivery of siRNA Targeting the Acid Ceramidase Gene for Cancer Therapy. Biomaterials 2011, 32, 3124− 3133. (50) Gong, J.; Chen, M.; Zheng, Y.; Wang, S.; Wang, Y. Polymeric Micelles Drug Delivery System in Oncology. J. Controlled Release 2012, 159, 312−323. (51) Kataoka, K.; Harada, A.; Nagasaki, Y. Block Copolymer Micelles for Drug Delivery: Design, Characterization and Biological Significance. Adv. Drug Delivery Rev. 2001, 47, 113−131. (52) Rösler, A.; Vandermeulen, G. W. M.; Klok, H. A. Advanced Drug Delivery Devices via Self-Assembly of Amphiphilic Block Copolymers. Adv. Drug Delivery Rev. 2012, 64, 270−279. (53) Gaspar, V. M.; Gonçalves, C.; de Melo-Diogo, D.; Costa, E. C.; Queiroz, J. A.; Pichon, C.; Sousa, F.; Correia, I. J. Poly(2-ethyl-2oxazoline)−PLA-g−PEI Amphiphilic Triblock Micelles for CoDelivery of Minicircle DNA and Chemotherapeutics. J. Controlled Release 2014, 189, 90−104. (54) Huang, H. Y.; Kuo, W. T.; Chou, M. J.; Huang, Y. Y. CoDelivery of Anti-Vascular Endothelial Growth Factor siRNA and Doxorubicin by Multifunctional Polymeric Micelle for Tumor Growth Suppression. J. Biomed. Mater. Res., Part A 2011, 97, 330−338. (55) Hagerman, P. J. Investigation of the Flexibility of DNA using Transient Electric Birefringence. Biopolymers 1981, 20, 1503−1535. (56) Kayitmazer, A. B.; Seyrek, E.; Dubin, P. L.; Staggemeier, B. A. Influence of Chain Stiffness on the Interaction of Polyelectrolytes with Oppositely Charged Micelles and Proteins. J. Phys. Chem. B 2003, 107, 8158−8165. (57) Ulrich, S.; Laguecir, A.; Stoll, S. Complexation of a Weak Polyelectrolyte with a Charged Nanoparticle. Solution Properties and Polyelectrolyte Stiffness Influences. Macromolecules 2005, 38, 8939− 8949. (58) Wallin, T.; Linse, P. Monte Carlo Simulations of Polyelectrolytes at Charged Hard Spheres with Different Numbers of Polyelectrolyte Chains. J. Chem. Phys. 1998, 109, 5089. (59) Akinchina, A.; Linse, P. Monte Carlo Simulations of Polyion− Macroion Complexes. 1. Equal Absolute Polyion and Macroion Charges. Macromolecules 2002, 35, 5183−5193. (60) Akinchina, A.; Linse, P. Monte Carlo Simulations of Polyion− Macroion Complexes. 2. Polyion Length and Charge Density Dependence. J. Phys. Chem. B 2003, 107, 8011−8021.

(61) Wang, D.; Wang, T.; Liu, J.; Yu, H.; Jiao, S.; Feng, B.; Zhou, F.; Fu, Y.; Yin, Q.; Zhang, P.; et al. Acid-Activatable Versatile Micelleplexes for PD-L1 Blockade-Enhanced Cancer Photodynamic Immunotherapy. Nano Lett. 2016, 16, 5503−5513. (62) Gary, D. J.; Lee, H.; Sharma, R.; Lee, J.-S.; Kim, Y.; Cui, Z. Y.; Jia, D.; Bowman, V. D.; Chipman, P. R.; Wan, L.; et al. Influence of Nano-Carrier Architecture on in Vitro siRNA Delivery Performance and in Vivo Biodistribution: Polyplexes vs Micelleplexes. ACS Nano 2011, 5, 3493−3505. (63) Sun, T.; Du, J.; Yao, Y.; Mao, C.; Dou, S.; Huang, S.; Zhang, P.Z.; Leong, K. W.; Song, E.-W.; Wang, J. Simultaneous Delivery of siRNA and Paclitaxel via a “Two-in-One” Micelleplex Promotes Synergistic Tumor Suppression. ACS Nano 2011, 5, 1483−1494. (64) Rothemund, P. W. K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440, 297−302. (65) Goddard, N. L.; Bonnet, G.; Krichevsky, O.; Libchaber, A. Sequence Dependent Rigidity of Single Stranded DNA. Phys. Rev. Lett. 2000, 85, 2400−2403. (66) Rad, S. M. A. H.; Bamdad, T.; Sadeghizadeh, M.; Arefian, E.; Lotfinia, M.; Ghanipour, M. Transcription Factor Decoy against Stem Cells Master Regulators, Nanog and Oct-4: a Possible Approach for Differentiation Therapy. Tumor Biol. 2015, 36, 2621−2629. (67) Sarwar, U. N.; Costner, P.; Enama, M. E.; Berkowitz, N.; Hu, Z.; Hendel, C. S.; Sitar, S.; Plummer, S.; Mulangu, S.; Bailer, R. T.; et al. Safety and Immunogenicity of DNA Vaccines Encoding Ebolavirus and Marburgvirus Wild-Type Glycoproteins in a Phase I Clinical Trial. J. Infect. Dis. 2015, 211, 549−557. (68) Sprouse, D.; Jiang, Y.; Laaser, J. E.; Lodge, T. P.; Reineke, T. M. Tuning Cationic Block Copolymer Micelle Size by pH and Ionic Strength. Biomacromolecules 2016, 17, 2849−2859. (69) Alhoranta, A. M.; Lehtinen, J. K.; Urtti, A. O.; Butcher, S. J.; Aseyev, V. O.; Tenhu, H. J. Cationic Amphiphilic Star and Linear Block Copolymers: Synthesis, Self-Assembly, and in Vitro Gene Transfection. Biomacromolecules 2011, 12, 3213−3222. (70) Synatschke, C. V.; Schallon, A.; Jérôme, V.; Freitag, R.; Müller, A. H. E. Influence of Polymer Architecture and Molecular Weight of Poly(2-(dimethylamino)ethyl methacrylate) Polycations on Transfection Efficiency and Cell Viability in Gene Delivery. Biomacromolecules 2011, 12, 4247−4255. (71) van de Wetering, P.; Moret, E. E.; Schuurmans-Nieuwenbroek, N. M. E.; van Steenbergen, M. J.; Hennink, W. E. Structure−Activity Relationships of Water-Soluble Cationic Methacrylate/Methacrylamide Polymers for Nonviral Gene Delivery. Bioconjugate Chem. 1999, 10, 589−597. (72) Plamper, F. A.; Ruppel, M.; Schmalz, A.; Borisov, O.; Ballauff, M.; Müller, A. H. E. Tuning the Thermoresponsive Properties of Weak Polyelectrolytes: Aqueous Solutions of Star-Shaped and Linear Poly(N,N -dimethylaminoethyl Methacrylate). Macromolecules 2007, 40, 8361−8366. (73) Car, A.; Baumann, P.; Duskey, J. T.; Chami, M.; Bruns, N.; Meier, W. pH-Responsive PDMS- b -PDMAEMA Micelles for Intracellular Anticancer Drug Delivery. Biomacromolecules 2014, 15, 3235−3245. (74) Agarwal, A.; Unfer, R.; Mallapragada, S. K. Novel Cationic Pentablock Copolymers as Non-viral Vectors for Gene Therapy. J. Controlled Release 2005, 103, 245−258. (75) Reinhardt, M.; Dzubiella, J.; Trapp, M.; Gutfreund, P.; Kreuzer, M.; Gröschel, A. H.; Müller, A. H. E.; Ballauff, M.; Steitz, R. FineTuning the Structure of Stimuli-Responsive Polymer Films by Hydrostatic Pressure and Temperature. Macromolecules 2013, 46 (16), 6541−6547. (76) Jakeš, J. Regularized Positive Exponential Sum (REPES) Program - A Way of Inverting Laplace Transform Data Obtained by Dynamic Light Scattering. Collect. Czech. Chem. Commun. 1995, 60, 1781−1797. (77) Hagerman, P. Flexibility of DNA. Annu. Rev. Biophys. Biomol. Struct. 1988, 17, 265−286. (78) Lu, Y.; Weers, B.; Stellwagen, N. C. DNA Persistence Length Revisited. Biopolymers 2002, 61, 261−275. 6719


Article

The Journal of Physical Chemistry B (79) Hammermann, M.; Steinmaier, C.; Merlitz, H.; Kapp, U.; Waldeck, W.; Chirico, G.; Langowski, J. Salt Effects on the Structure and Internal Dynamics of Superhelical DNAs Studied by Light Scattering and Brownian Dynamics. Biophys. J. 1997, 73, 2674−2687. (80) Langowski, J. Salt Effects on Internal Motions of Superhelical and Linear pUC8 DNA. Biophys. Chem. 1987, 27, 263−271. (81) Langowski, J.; Giesen, U. Configurational and Dynamic Properties of Different Length Superhelical DNAs Measured by Dynamic Light Scattering. Biophys. Chem. 1989, 34, 9−18. (82) Tricot, M. Comparison of Experimental and Theoretical Persistence Length of Some Polyelectrolytes at Various Ionic Strengths. Macromolecules 1984, 17, 1698−1704. (83) Izumrudov, V. A.; Wahlund, P. O.; Gustavsson, P. E.; Larsson, P. O.; Galaev, I. Y. Factors Controlling Phase Separation in Water−Salt Solutions of DNA and Polycations. Langmuir 2003, 19, 4733−4739. (84) Shovsky, A.; Varga, I.; Makuska, R.; Claesson, P. M. Formation and Stability of Water-Soluble, Molecular Polyelectrolyte Complexes: Effects of Charge Density, Mixing Ratio, and Polyelectrolyte Concentration. Langmuir 2009, 25, 6113−6121. (85) Wang, Y.; Kimura, K.; Dubin, P. L.; Jaeger, W. PolyelectrolyteMicelle Coacervation: Effects of Micelle Surface Charge Density, Polymer Molecular Weight, and Polymer/Surfactant ratio. Macromolecules 2000, 33, 3324−3331. (86) Bakeev, K. N.; Izumrudov, V. A.; Kuchanov, S. I.; Zezin, A. B.; Kabanov, V. A. Kinetics and Mechanism of Interpolyelectrolyte Exchange and Addition Reactions. Macromolecules 1992, 25, 4249− 4254. (87) Lokitz, B. S.; Convertine, A. J.; Ezell, R. G.; Heidenreich, A.; Li, Y.; McCormick, C. L. Responsive Nanoassemblies via Interpolyelectrolyte Complexation of Amphiphilic Block Copolymer Micelles. Macromolecules 2006, 39, 8594−8602. (88) Sukhishvili, S. A.; Kharlampieva, E.; Izumrudov, V. Where Polyelectrolyte Multilayers and Polyelectrolyte Complexes Meet. Macromolecules 2006, 39, 8873−8881. (89) Laaser, J. E.; Jiang, Y.; Sprouse, D.; Reineke, T. M.; Lodge, T. P. pH- and Ionic-Strength-Induced Contraction of Polybasic Micelles in Buffered Aqueous Solutions. Macromolecules 2015, 48, 2677−2685. (90) Petrov, A. I.; Antipov, A. A.; Sukhorukov, G. B. Base−Acid Equilibria in Polyelectrolyte Systems: From Weak Polyelectrolytes to Interpolyelectrolyte Complexes and Multilayered Polyelectrolyte Shells. Macromolecules 2003, 36, 10079−10086. (91) Choi, J.; Rubner, M. F. Influence of the Degree of Ionization on Weak Polyelectrolyte Multilayer Assembly. Macromolecules 2005, 38, 116−124. (92) Burke, S. E.; Barrett, C. J. Acid-Base Equilibria of Weak Polyelectrolytes in Multilayer Thin Films. Langmuir 2003, 19, 3297− 3303. (93) Laguecir, A.; Stoll, S. Adsorption of a Weakly Charged Polymer on an Oppositely Charged Colloidal Particle: Monte Carlo Simulations Investigation. Polymer 2005, 46, 1359−1372. (94) Samokhina, L.; Schrinner, M.; Ballauff, M.; Drechsler, M. Binding of Oppositely Charged Surfactants to Spherical Polyelectrolyte Brushes: A Study by Cryogenic Transmission Electron Microscopy. Langmuir 2007, 23, 3615−3619. (95) Benoit, H.; Froelich, D. In Light Scattering from Polymer Solutions; Huglin, M. B., Ed.; Academic Press: London and New York, 1972; Chapter 9. (96) Boustta, M.; Leclercq, L.; Vert, M.; Vasilevskaya, V. V. Experimental and Theoretical Studies of Polyanion−Polycation Complexation in Salted Media in the Context of Nonviral Gene Transfection. Macromolecules 2014, 47, 3574−3581. (97) Skepö, M.; Linse, P. Dissolution of a Polyelectrolyte-Macroion Complex by Addition of Salt. Phys. Rev. E: Stat. Phys., Plasmas, Fluids, Relat. Interdiscip. Top. 2002, 66, 051807. (98) Trinh, C. K.; Schnabel, W. Ionic Strength Dependence of the Stability of Polyelectrolyte Complexes. Its Importance for the Isolation of Multiply Charged Polymers. Angew. Makromol. Chem. 1993, 212, 167−179.

(99) Kovacevic, D.; Van der Burgh, S.; De Keizer, A.; Cohen Stuart, M. A. Kinetics of Formation and Dissolution of Weak Polyelectrolyte Multilayers: Role of Salt and Free Polyions. Langmuir 2002, 18, 5607− 5612. (100) Antila, H. S.; Sammalkorpi, M. Polyelectrolyte Decomplexation via Addition of Salt: Charge Correlation Driven Zipper. J. Phys. Chem. B 2014, 118, 3226−3234. (101) Jonsson, M.; Linse, P. Polyelectrolyte−Macroion Complexation. II. Effect of Chain Flexibility. J. Chem. Phys. 2001, 115, 10975. (102) Mengarelli, V.; Auvray, L.; Pastré, D.; Zeghal, M. Charge Inversion, Condensation and Decondensation of DNA and Polystyrene Sulfonate by Polyethylenimine. Eur. Phys. J. E: Soft Matter Biol. Phys. 2011, 34, 127. (103) Boddohi, S.; Moore, N.; Johnson, P. A.; Kipper, M. J. Polysaccharide-Based Polyelectrolyte Complex Nanoparticles from Chitosan, Heparin, and Hyaluronan. Biomacromolecules 2009, 10, 1402−1409. (104) Chen, H.; Meisburger, S. P.; Pabit, S. A.; Sutton, J. L.; Webb, W. W.; Pollack, L. Ionic Strength-Dependent Persistence Lengths of Single-Stranded RNA and DNA. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 799−804.

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