Complexation of DNA with Cationic Copolymer Micelles: Effects of

Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

Complexation of DNA with Cationic Copolymer Micelles: Effects of DNA Length and Topology Yaming Jiang,† Theresa M. Reineke,*,‡ and Timothy P. Lodge*,†,‡ †

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

ABSTRACT: Micelleplexes are promising gene delivery vehicles that form when DNA complexes with polycationic micelles. In this study, the influence of DNA length and topology on the structure and colloidal stability of micelleplexes was explored using a model system. The cationic micelles were composed of poly(2-(dimethylamino)ethyl methacrylate)-blockpoly(n-butyl methacrylate) and were complexed with linear DNA and circular plasmids of 2442, 4708, and 7537 base pairs. The cationic micelles had a mean core radius of 8 ± 1 nm and a mean hydrodynamic radius of 34 ± 1 nm in buffer at pH 5 and 100 mM ionic strength. The formation of micelleplexes was monitored by turbidimetric titration as a function of N/P ratio (amine in micelle corona/phosphate on DNA) in acetate buffers of various ionic strengths. The structure and size evolution of micelleplexes were studied by dynamic light scattering and cryo-TEM, while the composition of micelleplexes was estimated using static light scattering. The combination of these techniques revealed that increasing DNA length resulted in increased micelleplex size at N/P > 1; this was attributed to an increased propensity for longer DNA to bridge between micelles. At N/P < 1, however, longer DNA enhances the stability of micelleplexes against aggregation by providing additional steric repulsions between micelleplexes. At high ionic strength, increasing DNA length also shifts titration curves to higher N/P ratios as the structure of micelleplexes changes with DNA length. On the other hand, DNA topology showed minimal influence on the titration curves, structure, and long-term stability of micelleplexes. Overall, this work illustrates how polycationic micelles may serve as compaction agents for long chain DNA and how factors such as DNA length can be used to tune the structure and colloidal stability of micelleplexes.

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INTRODUCTION Interpolyelectrolyte complexes (IPEC) are formed when oppositely charged polyelectrolytes are combined and allowed to form ion pairs between charged groups.1,2 These materials are attractive for a variety of applications, including filtration,3,4 encapsulation,5,6 water treatment,7,8 and therapeutic delivery,9,10 due to their versatile chemical composition and environmental responsiveness. The main driving force for complexation is the entropic gain from liberating the counterions.11,12 Although the complexation between linear polyelectrolytes has been extensively studied13−15 for use in self-healing materials16 and surface coatings,17,18 the incorporation of polyelectrolytes with predefined structures, such as dendrimers, 19,20 spherical micelles, 21−24 or cylindrical brushes,25,26 opens the possibility to construct complexes of well-defined hierarchical structure,1,27 such as onion-like23 or multicompartment24 spheres or cylinders with helical topography.26 Furthermore, by manipulation of the structure and composition of the complexing polyelectrolyte, additional control of the structure, stability, and equilibration kinetics of the complexes can be achieved.21,28−30 The incorporation of prestructured polyelectrolytes therefore is an effective strategy in controlling the structures and properties of IPECs. © XXXX American Chemical Society

Recently, IPECs between DNA and prestructured polycations have received attention for their potential applications in gene delivery31−35 as well as for a fundamental understanding of the interactions between DNA and proteins.20,36,37 Theoretical studies37,38 and simulations39,40 have examined the complexation behavior between semiflexible polyanions and cationic spheres to understand the compaction of DNA in chromatin. Experimentally, polycations of predefined structures were also used to complex with DNA in order to construct delivery vehicles with controllable structures and desirable physical and biological properties.31,34,41,42 Among many potential platforms, complexes between DNA and selfassembled block polymer micelles, namely micelleplexes, have shown promise in gene silencing in vitro and tumor accumulation and inhibition in vivo31−33 and in codelivery with small molecule therapeutics.31,33 However, more physical studies of micelleplexes are needed, particularly for long chain DNA that are larger than the micelles, to correlate the structure and properties of micelleplexes to their individual components Received: October 13, 2017 Revised: January 13, 2018

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DOI: 10.1021/acs.macromol.7b02201 Macromolecules XXXX, XXX, XXX−XXX

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effectively complex with DNA, as a homopolymer or as part of an assembly.52−55 All DNAs used in this study were doublestranded, and the DNA topologies were either linear or plasmid (mostly supercoiled). The DNA length was varied from 2442 to 7537 base pairs (bp). The formation of micelleplexes was monitored by turbidimetric titration, and their structure and size evolution were probed by dynamic light scattering, static light scattering, and cryogenic transmission electron microscopy as functions of solution ionic strength and N/P ratio (total amine groups in micelle corona/total phosphate groups on DNA).

and preparation conditions and thereby to inform future design and optimization. Fundamentally, the compaction of semiflexible macromolecules, such as DNA, into a condensed structure, such as a sphere, remains as a significant challenge and a fascinating topic. Previous studies on micelleplexes and related systems have achieved understanding of micelleplex formation as a balance between thermodynamic driving forces and kinetic constraints, whereby the charge ratio and solution ionic strength play intricate roles.22,43 The complexation between DNA and cationic micelles is kinetically limited at low ionic strength. When micelles are in excess, micelleplexes are solubilized by excess positive charge in the micelle corona. When DNA is in excess, micelleplexes become overcharged, resulting from excess DNA at the micelle surface. In both cases, multiple-micelle complexes may form due to the association of neutralized patches on the micelle surface and/or the bridging of DNA between micelles. The kinetic structure of a micelleplex influences both its instantaneous solubility and titration diagram. Over time, the kinetic structures evolve toward thermodynamic equilibrium, which determines the long-term stability of the formulation. In addition, high solution ionic strength promotes the structural evolution of a micelleplex by facilitating rearrangement of ion pairing, but the path of evolution depends on the charge ratio. Annealed micelleplexes with micelles in excess are stable and show a tendency to evolve to more compact complexes, whereas micelleplexes with DNA in excess showed poor colloidal stability with increasing size and eventually precipitate. In addition to the solution ionic strength and the charge ratio, the stiffness of DNA has also been found to influence the properties of micelleplexes in comparison to flexible polyanions, which contributes to unique properties of micelleplexes. Double-stranded DNA is semiflexible with an intrinsic persistence length of about 50 nm.44,45 Simulation studies have shown that while flexible polyelectrolytes randomly absorb onto oppositely charged spheres, the condensation of stiff chains around the same spheres may result in more regular patterns.39,40,46 Experimentally, our group demonstrated that micelleplexes of long chain DNA have different titration curves, structures, equilibration kinetics, and tolerance to high ionic strength than complexes of the same cationic micelles with flexible polystyrenesulfonate of a similar total number of charge units.43 Despite recent progress, many factors that may affect micelleplex properties remain unexplored. For example, plasmids used in gene delivery are often composed of supercoiled DNA with tertiary topologies.47,48 Compared to linear DNA, the tertiary topology of supercoiled DNA could affect interactions with cationic micelles, as has been shown for selective linear polycations and nucleosome core particles.49,50 In addition, the structure and corona chemistry of the cationic micelles could also play an important role in modulating the colloidal stability and equilibrium kinetics of their complexes, as in the case with flexible polyanions.21,30 In this study, the influence of DNA length and topology on the structure and stability of complexes with cationic polymeric micelles was investigated. The cationic polymeric micelles were composed of poly(2-(dimethylamino)ethyl methacrylate)-block-poly(n-butyl methacrylate) (poly(DMAEMA)-b-poly(nBMA)) chains, which were previously thoroughly characterized as spherical micelles with core−shell structures that were kinetically trapped (i.e., no chain exchange).51 The poly(DMAEMA) corona block is a weak cationic polyelectrolyte that has been shown to

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MATERIALS AND METHODS

Materials. Dialysis tubing (cutoff Mw = 6−8 kDa) was purchased from Spectra/Por, treated with 0.1 wt % ethylenediaminetetraacetic acid (EDTA) solution, stored in ∼0.05 wt % sodium azide solution, and triple rinsed with Milli-Q water before use. Acetate buffers were prepared at pH 5 using acetic acid and sodium hydroxide, and ionic strength was controlled through the addition of sodium chloride. HpaI restriction enzyme and Quick-load Purple 2-Log DNA ladder (0.1− 10.0 kb) were purchased from New England Biolabs Inc. and used as received. Mixtures of phenol/chloroform/isoamyl alcohol (25:24:1 by volume) were purchased from VMR International and were adjusted to pH 8.0 with TRIS (tris(hydroxymethyl)aminomethane) buffer before use. Micelle Formation. Cationic micelles were formed by direct dissolution of poly(DMAEMA)-b-poly(nBMA) in deionized water at 1 mg/mL at room temperature. The molar masses of DMAEMA and nBMA blocks are 27 and 14 kDa, respectively, and the polymer dispersity is 1.10. The polymer synthesis and micelle characterization were described elsewhere.51 All micelle solutions were dialyzed against buffers of desired pH and ionic strength, and after dialysis the concentration was determined by UV−vis spectroscopy using the absorbance of the trithiocarbonate group as previously described.51 DNA Preparation. Plasmids of 2442, 4708, and 7537 bp were propagated in Escherichia coli cells and routinely purified using PerfectPrep Endofree Plasmid Maxi Kit (VWR, Radnor, PA). Linear DNAs were prepared by the digestion of plasmids using the HpaI restriction enzyme, which gives a single blunt break for all three plasmids. Complete digestion was confirmed by agarose gel electrophoresis. Only the band corresponding to the linear DNA was visible after digestion, as shown in Figure S1 of the Supporting Information. Linear DNA was then purified by chloroform−phenol extraction as previously described.43 Before use, DNA samples were dialyzed against Milli-Q water to remove excess salt, freeze-dried, and redissolved in buffers of desired pH and ionic strengths. The concentrations were determined by UV−vis spectroscopy using an extinction coefficient of 0.021 (μg/mL)−1 cm−1 at 260 nm. Turbidimetric Titrations. The titrations were carried out as previously described.43 Briefly, DNA and micelles solutions were prepared at 0.2 mg/mL unless otherwise specified. In a typical titration, DNA (micelle) solution was added to a 2 mL micelle (DNA) solution at a rate of 40 μL/min while stirring at 500 rpm. After every 15 additions, 600 μL of solution was taken out so that a range of N/P ratios can be reached during a titration with a reasonable total number of additions. The total mass concentration was maintained at 0.2 mg/ mL unless otherwise specified. The solution transmittance was monitored using a custom-built setup with a HeNe laser (632 nm wavelength) and a laser power meter (Spex Industries). The transmitted intensity was read between two additions, and the homogeneity of the solution was visually inspected after each addition. Micelleplexes were prepared in the same fashion, except that the titrations were stopped at targeted N/P ratios. The minority component (in terms of N/P ratio) was always titrated into the majority so that the target N/P ratios were reached sooner. Dynamic Light Scattering. Dynamic light scattering (DLS) was conducted using a Brookhaven Instrument BI-200DM multiangle light-scattering instrument with a 637 nm laser. All measurements B


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Macromolecules were performed at 23 °C, and for each sample correlation functions were collected at five angles. All vials that contained samples for DLS measurements were triple rinsed with Milli-Q water and acetone and dried while covered in aluminum foil to minimize the contribution of dust. Before measurements, all micelle samples were filtered through 0.2 μm filters and all DNA samples were filtered through 0.45 μm filters. All solutions were filtered before complexation only, to avoid potential impact on micelleplex structures. For calculations of hydrodynamic radii, the viscosity and refractive index of the buffers were estimated as those of sodium chloride solutions of the same ionic strength. For DNA samples, correlation functions were fitted using a doubleexponential model, following previous literature.56−58 The center-ofmass diffusion coefficients of DNA were obtained by extrapolating the diffusion coefficients of the slower mode to the limit of zero scattering vector and were averaged over three ionic strengths (20, 100, and 500 mM), as Dt is insensitive to solution ionic strength within this range.56,57 For micelleplex samples, the correlation functions were fitted to either a second cumulant or double-exponential model, depending on the size distribution, which was assessed by applying the REPES algorithm to the correlation function acquired at 90°.59 More fitting details are shown in the Supporting Information. Since the light scattering signal is weighted toward larger particles, the presence of free DNA molecules was unlikely to be captured by DLS when micelleplexes were also present in the solution. Static Light Scattering. Static light scattering (SLS) was conducted using the same Brookhaven Instrument as DLS and sample preparation followed the same cleaning protocol to reduce the contribution of dust. For SLS measurements, micelleplexes at N/P = 5 were prepared by titration at 0.2 mg/mL and then diluted to lower concentrations immediately before measurements. Micelleplexes with plasmid DNA of 7537 bp at 20 mM ionic strength were measured at day 1, and others were measured during a time period when the size distributions of the micelleplexes remained constant. Cryogenic Transmission Electron Microscopy. Cryogenic transmission electron microscopy (cryo-TEM) was carried out using a FEI Tecnai G2 Spirit BioTWIN microscope coupled with a CCD camera (2048 × 2048 pixels) and a single-tilt cryo holder. The microscope was operating at 120 kV, and images were captured at underfocus for adequate contrast. The solution samples were vitrified using a Vitrobot (FEI) as previously described.43 Briefly, lacy carbon/ Formvar grids (Ted Pella, 300 mesh) were treated using a PELCO easiGlow glow discharge cleaning system and then transferred into the climate-controlled chamber of the Vitrobot, which was controlled at 26 °C with ∼98% humidity. Inside the chamber, each grid was deposited with 2.5−3.2 μL sample solution, blotted for 5 s, rested for 1 s, and then plunged into liquid ethane surrounded by liquid nitrogen. The vitrified samples were subsequently transferred to and stored in liquid nitrogen until imaging. The micelleplexes were vitrified either at day 1 or at a time point when the hydrodynamic radii of the micelleplexes were independent of time.

linear or “SC” for plasmid [supercoiled]) and length (2442, 4708, or 7537 bp), as shown in Table 1. The topology of DNA Table 1. Selected Properties of DNAs Used abbreviation

conformation

DNA/micelle charge ratioa

L2442 L4708 L7537 SC2442 SC4708 SC7537

linear linear linear supercoiledb supercoiledb supercoiledb

0.3 0.6 1.0 0.3 0.6 1.0

Dtc (×10−12 m2/s)

Rh (nm) at 100 mM

3.8 2.5 2.1 5.2 3.2 2.4

61 92 111 44 71 95

a

Calculated as the charge ratio between one DNA molecule and one micelle. bA majority of plasmids are supercoiled, with small percentages of open circular or linear forms. cDt were measured in acetate buffers at pH 5 and 20, 100, and 500 mM ionic strength.

was confirmed by gel electrophoresis as shown in Figure S1, and the hydrodynamic radii of DNA chains were calculated from the diffusion coefficients measured by DLS. The centerof-mass diffusion coefficient Dt of DNA scales with the molar mass M such that Dt ∼ Mν. For supercoiled DNA ν = −0.7 ± 0.1, and for linear DNA ν = −0.5 ± 0.1. These scaling exponents agree within error with the literature value and theoretical predictions.56 More fitting details are shown in the Supporting Information (Table S1 and Figure S2). As Table 1 shows, the supercoiled DNA chains have smaller hydrodynamic radii than the linear chains of the same length. With increasing DNA length, the hydrodynamic radius of DNA chains increases and the charge ratio between one DNA molecule to one micelle also increases from 0.3 to 1.0. It is important to note that the hydrodynamic radii of the DNA chains are generally larger than those of the micelles. Turbidimetric Titrations. The complexation process between DNA and the cationic micelles as a function of N/P ratio was studied by turbidimetric titration at various ionic strengths. For example, Figure 1 shows the titration curves

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RESULTS Micelles and DNA Library. The cationic micelles used in this study were characterized previously using cryo-TEM and light scattering (dynamic and static) and were found to be spherical and kinetically constrained with respect to chain exchange at room temperature.51 These micelles have a core radius of 8 ± 1 nm and a hydrodynamic radius of 34 ± 1 nm in pH 5 buffer at 100 mM ionic strength. The molar mass of these micelles measured by static light scattering is 3.8 ± 0.4 MDa, which corresponds to (1.6 ± 0.2) × 104 amine groups per micelle. In this study, the solution pH was kept at 5 so that almost all amine groups were charged during complexation. The solution ionic strength was varied between 20 and 500 mM to investigate its influence on micelleplex structure and stability. A library of DNA samples was prepared for complexation. For each DNA, its abbreviation denotes its topology (“L” for

Figure 1. Turbidimetric titration curves of micelles with SC2442 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. C


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Macromolecules between micelles and SC2442 in pH 5 buffer of 20 mM ionic strength. The titration solutions started as homogeneous, as shown by the filled symbols in Figure 1. With increasing amount of DNA (micelles) added into micelles (DNA) solutions, the solution transmittance decreased and soon precipitated after the transmittance decreased below 0.9. This is a common feature shared among titrations between polyelectrolyte micelles and oppositely charged linear polyelectrolytes.21,22,43 Particularly in this case, the inhomogeneous region was entirely at N/P < 1, whereas for many inter(poly)electrolyte systems it is centered on N/P ∼ 1.21,22 This asymmetry of titration curves shown in Figure 1 was also observed between L2442 and the same micelles and was attributed to the stiffness of the DNA, as detailed in our previous work.43 Because of the asymmetry of size and geometry between the cationic micelle and DNA chains, the initial complexation process differs depending on the titration direction. With micelles in excess, added DNA chains could interact with multiple micelles and form multiple-micelle complexes with micelles decorating the DNA chains. With DNA in excess, on the other hand, added micelles could interact with multiple DNA chains and form single-micelle complexes with excess DNA chains as coronas. Eventually, aggregation among primary complexes happens when the added component bridges between complexes, which leads to formation of large aggregates and decrease in solution transmittance. To investigate the influence of DNA length, supercoiled DNAs of three lengths were titrated with micelles in pH 5 buffers at 20, 100, and 500 mM ionic strength. To aid in comparison among titrations, the N/P ratio at which the solution transmittance first decreased below 0.9 is defined as the titration end point, as plotted in Figure 2. As Figure 2 shows, a titration diagram was obtained for each DNA length with the areas above the top curve and below the bottom curve corresponding to homogeneous regions with one component in excess and areas in between the two curves corresponding to inhomogeneous regions. With increasing solution ionic strength, the inhomogeneous regions generally widened, which is similar to what has been observed with other polyelectrolyte systems.22,60 However, the direction of inhomogeneous expansion depended on DNA length. Specifically, the inhomogeneous regions of SC2442 expanded toward N/P < 1, whereas the inhomogeneous regions of SC7537 expanded more significantly toward N/P > 1 with increasing ionic strength. This suggests that the structures of micelleplexes of different DNA length could be organized differently, thereby contributing to different colloidal stability at high ionic strength. The location of the inhomogeneous region also depends on DNA length when a different micelle with more total amine groups51 was titrated, as shown for comparison in Figure 2a,c. This indicates that the expansion of the inhomogeneous region does not depend on the charge ratio between one DNA molecule and one micelle. By reduction of the concentration of the titration solution from 0.2 to 0.04 mg/ mL, the expansion of the inhomogeneous region at high ionic strength was reduced. This is expected as flocculation is a kinetic process that is retarded by reducing the solution concentration.61 However, the dependence on DNA length was also reduced at the lower concentration, as the reduction of inhomogeneous region is asymmetric and is primarily in the direction opposite to the influence of DNA length. This

Figure 2. Titration end points of micelles with (a) SC2442, (b) SC4708, and (c) SC7537 as a function of solution ionic strength. Red symbols (2) are titration end points between the same DNA with (14−23) micelles, which have more amine groups per micelle. Properties of (14−23) micelles are summarized in Table S2. Cyan symbols (3) show titration end points between the same DNA and (27−14) micelles at a lower concentration (0.04 mg/mL). The original titration curves are shown in Figures S4, S6, S8, and S10−S13.

suggests that the influence of DNA length on titration diagram depends on the concentration of the complexing components. In comparison to DNA length, the topology of DNA showed minimal influence on the titration diagrams; linear and supercoiled DNA are compared in Figure 3, where titration end points are plotted as a function of DNA length. Figure 3b again captures the general trends of titration diagram for supercoiled DNA: with increasing solution ionic strength, the inhomogeneous region expands; at high ionic strength, the D


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Figure 3. Titration end points of micelles with (a) linear and (b) supercoiled DNA as a function of DNA chain length. The original titration curves for linear DNA are shown in Figures S3, S5, and S7. Selected error bars are shown, which were calculated from the duplicate titrations shown in Figure S9. Schematic illustrations of homogeneous and inhomogeneous regions are shown for titrations at 20 mM ionic strength.

close to the rest of the population that the REPES algorithm could not distinguish a separate population. This suggests that longer DNA forms larger multiple-micelle complexes than micelleplexes of shorter DNA. With increasing ionic strength, as shown in Figure 5b, multiple-micelle complexes of longer DNA remained larger than that of shorter DNA. For both DNA lengths, micelleplexes remained soluble and showed a tendency to rearrange into smaller complexes over time, which is in agreement with previous studies.22,43 These micelleplexes with micelles in excess are also stable after dialysis into phosphate buffered saline (PBS buffer) of pH 7.4 and 154 mM ionic strength. The size distributions of these micelleplexes showed little changes after this buffer exchange, as shown in Figure S32. With DNA in excess (N/P = 0.2), increasing DNA length enhanced the colloidal stability of micelleplexes. With shorter DNA, micelleplexes showed a single size distribution initially, and their size increased over time, resulting in precipitation after 14 days, as shown in Figure 5c. This behavior agrees with previous studies in that complexes with polyanions in excess have poor long-term colloidal stability.22,43 In comparison, micelleplexes of longer DNA showed bimodal size distributions that were stable over at least a month. This suggests that increasing DNA length might retard the structural evolution of micelleplexes and therefore improve their colloidal stability when DNA is in excess. In both cases (N/P = 5 and 0.2), DNA topology did not affect the long-term stability of micelleplexes. This agrees with the previous hypothesis from turbidimetric titrations that the topology of DNA does not significantly affect the colloidal stability of the micelleplexes. By changing topology from supercoiled to a linear form, the resulting micelleplex size showed a slight increase, as shown in Figure 5b,c. Since linear DNA has a larger hydrodynamic radius than its supercoiled form, it is plausible that linear DNA might contribute more to the overall size of micelleplexes if the structural organization is similar between micelleplexes of linear and supercoiled DNA. Static Light Scattering. By use of static light scattering, the composition of micelleplexes with micelles in excess was estimated, which confirms that longer DNA forms larger complexes by incorporating more micelles into a given complex. Table 2 shows the molar mass of micelleplexes measured by static light scattering (SLS) at N/P = 5. The global stoichiometric number ratios between micelles and DNA chains (#micelle/#DNA) at N/P = 5 were also calculated for each

inhomogeneous region expands to lower N/P ratios for shorter DNA but to higher N/P ratios for longer DNA. Figure 3a shows the titration diagram of linear DNA, which exhibits the same dependence on DNA length and solution ionic strength. This similarity suggests that the topology of DNA does not significantly affect the organization of micelleplexes or the stability and aggregation tendency of the micelleplexes. Dynamic Light Scattering. The influence of DNA length and topology on the size and stability of micelleplexes was examined by DLS, which shows that DNA length plays an important role in determining the size distribution and longterm colloidal stability of micelleplexes, whereas DNA topology only has a secondary influence. Since previous work has shown that the structural evolution and long-term stability of micelleplexes depend on the identity of the species in excess,22,43 the influence of DNA length was analyzed first for N/P > 1 (micelles in excess) and then for N/P < 1 (DNA in excess). With micelles in excess (N/P = 5), increasing DNA length increased the size of multiple-micelle complexes. As shown in Figures 4 and 5a, micelleplexes prepared using longer DNA

Figure 4. Size distributions of micelleplexes at N/P = 5 in acetate buffer of pH 5 and 20 mM ionic strength at day 1.

showed bimodal size distributions, with one population of a similar size as single micelles (Rh ≈ 40 nm) and one population of large complexes corresponding to multiple-micelle complexes (Rh ≈ 90 nm). In comparison, micelleplexes with shorter DNA formed micelleplexes of a monomodal size distribution with a similar size as the micelles. Since micelles are in excess, this population may consist of excess micelles, single-micelle complexes, and multiple-micelle complexes whose size is so E


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estimated with additional assumptions. First, it is assumed that all micelles are complexed. Second, for SC2442 micelleplexes, complexes are assumed to have uniform compositions, as they reach stable monomodal size distributions as shown in Figure 5. This assumption leads to the estimates that one SC2442 micelleplex on average contains 2.8 ± 0.5 micelles and 1.8 ± 0.3 DNA chains at 20 mM ionic strength. With increasing solution ionic strength, the average numbers of micelles and DNA chains per micelleplexes increase slightly. For SC7537 micelleplexes, two populations of uniform distributions were assumed, as the micelleplexes showed bimodal size distributions. In addition, each complex was assumed to contain only one DNA chain, as the number of micelles is 5 times the number of DNA chains at N/P = 5. Those assumptions lead to the estimates that SC7537 micelleplexes have one population of smaller complexes (population I), which on average contains three micelles and one DNA chain, and one population of larger complexes (population II), which on average contains tens of micelles around one DNA chain. The number fraction of population II increases with solution ionic strength, which shows that as electrostatic interactions become more screened, formation of larger multiple-micelle complexes becomes more favored. Upon comparison of the composition of SC2442 micelleplexes with population II of SC7537 micelleplexes, it is also clear that increasing DNA chain length favors formation of large multiplemicelle complexes as one DNA chain is more likely to bridge between a greater number of micelles. It is worth noting that these estimates of micelleplex composition rely on assumptions about micelleplex populations and do not consider the inevitable dispersity of the micelleplexes. As Figure 4 shows that micelleplexes formed have a range of hydrodynamic radii, their compositions should vary around the averaged value and might include situations where the assumptions fail. For example, free micelles could coexist with micelleplexes as the micelles are in excess at N/P = 5. It is also possible that SC7537 micelleplexes can have more than one DNA chain per complex. Cryo-TEM. To complement the DLS and SLS results, micelleplexes structures were visualized by cryo-TEM as Figure 6 illustrates how DNA length affects the structure of micelleplexes at different N/P ratios. In Figure 6, micelleplexes were imaged either at day 1 or in a period where the size distribution of micelleplexes stayed constant. Therefore, these images represent the initial kinetic structures of micelleplexes. With micelles in excess (N/P = 5), micelleplexes of longer DNA formed larger complexes by incorporating more micelles into one complex. The micelle cores are easily identifiable in the cryo-TEM images, while DNA molecules have weak contrast and are only visualized in selective images, as shown in Figure 7, which shows that DNA chains bridge between micelles to form multiple-micelle complexes. The formation of multiple-micelle complexes can also be inferred from the close association of two or more micelles, since micelles would otherwise be more randomly dispersed.51 As Figure 6a,b shows, micelleplexes at 20 mM ionic strength are composed of a significant fraction of free micelles or single-micelle complexes in addition to multiple-micelle complexes. Compared to micelleplexes of SC2442, multiple-micelle complexes were more prevalent in micelleplexes of SC7537, which agrees with the DLS result that micelleplexes of SC7537 had bimodal size distributions whereas micelleplexes of SC2442 had a single size distribution. With increasing solution ionic strength, the

Figure 5. Hydrodynamic radii of micelleplexes in acetate buffers of pH 5 with solution ionic strength of (a, c) 20 mM and (b) 500 mM at (a, b) N/P = 5 and (c) N/P = 0.2. For samples with bimodal size distributions, the hydrodynamic radii of both populations are shown. The cross symbols “×” indicate the observation of precipitation, and the corresponding hydrodynamic radii were measured on particles that remained in the upper clear portion of the solutions. The size distributions of the micelleplexes obtained by applying a REPES algorithm are shown in Figures S15−S18, and the values of their hydrodynamic radii are tabulated in Tables S4−S7.

measured system, as shown in Table 2. Using the molar mass of micelleplexes and the known concentrations of micelles and DNA chains, the compositions of micelleplexes can be F


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Table 2. Static Light Scattering Measurements on Micelleplexes at N/P = 5 in Acetate Buffers and Estimated Micelleplex Compositionsa SC2442 (#micelle/#DNA = 1.6)

SC7537 (#micelle/#DNA = 5.0)

ionic strength (mM) Mw (MDa) Rg (nm) estimated compositionsb (number fraction) #micelle/complex #DNA/complex

20 14 ± 2 40 ± 6 population I 2.8 ± 0.5 1.8 ± 0.3

20 39 ± 5 90 ± 10

ionic strength (mM) Mw (MDa) Rg (nm) estimated compositionsb (number fraction) #micelle/complex #DNA/complex

500 19 ± 2 69 ± 9 population I 4.0 ± 0.7 2.5 ± 0.4

population I (∼86%) 3 1c

population II (∼14%) 13−24 1c 500 31 ± 4 120 ± 20

population I (∼75%) 3 1c

population II (∼25%) 8−16 1c

a # denotes the number of the particles. #micelle/#DNA is the global stoichiometric number ratio between micelle and DNA chains. bBest estimations that fulfill Mw measured by SLS and number ratio between micelles and DNA chains. Assumptions, calculations, and additional supporting evidence are detailed in the Supporting Information. cThe number of SC7537 chain per complex is fixed as 1.

Figure 7. Selective cryo-TEM images of micelleplexes where DNA chains were visualized. These micelleplexes were prepared in acetate buffer at pH 5 and 500 mM ionic strength, with N/P = 5.

partially neutralized complexes due to hydrophobic interactions. This hypothesis also explains the dependence of micelleplex size on DNA chain length, as longer DNA chains are more likely to bridge micelles and form larger multiplemicelle complexes. With DNA in excess, micelleplexes of SC2442 were all large aggregates, as shown in Figure 6e, whereas micelleplexes of SC7537 showed two distinct populations, as shown in Figure 6f, with one of large aggregates and one of small complexes of similar size as single micelles. This observation agrees well with DLS results and shows that micelleplexes of longer DNA can sustain a population of small complexes, whereas micelleplexes of shorter DNA aggregated into large complexes that eventually led to precipitation. In comparison, DNA topology did not exert a noticeable influence on the micelleplex structure. In Figure S19, representative images are shown for micelleplexes of linear DNA that were prepared in the same conditions as micelleplexes of supercoiled DNA shown in Figure 6. At each condition, micelleplexes of linear DNA shared the same

Figure 6. Cryo-TEM images of micelleplexes formed with SC2442 and SC7537 in pH 5 buffers of various ionic strength and N/P ratios. The scale bar is 200 nm, and the red arrows on the second row point to multiple-micelle complexes. Micelleplexes at 500 mM ionic strength and N/P = 0.2 were imaged at day 1. Cryo-TEM images of micelleplexes of linear DNA formed under the same conditions are shown in Figure S19, and additional representative images are shown in Figures S20−S31.

number of SC7537 multiple-micelle complexes increased significantly (see Figure 6d), which agrees with the SLS results. Since DNA chains are semiflexible and have contour lengths much longer than the micelle radius, the formation of multiplemicelle complexes is most likely predominantly caused by DNA bridging between multiple micelles, rather than association of G


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small micelleplexes from aggregation, and therefore two distinct populations were captured in DLS and cryo-TEM. DNA length affects the instantaneous colloidal stability of micelleplexes, which is reflected in the titration diagrams (Figures 2 and 3). During a titration, the reduction of solution transmittance indicates a size increase of micelleplexes, which eventually leads to precipitation. When DNA is titrated into micelle solutions at high ionic strength, micelleplexes precipitate sooner with longer DNA chains. This is due to the fact that longer DNA chains are more likely to bridge between multiple micelles and form large micelleplexes, as shown by SLS. Such flocculation by bridging was also reported previously, which suggests that bridging interactions of high molecular weight polyelectrolytes can reduce the colloidal stability of charged particles and promote the onset of flocculation.62−65 When micelles are titrated into DNA solutions, titration curves of longer DNA are less influenced by solution ionic strength as the micelleplexes are stabilized by steric repulsion, in addition to electrostatic repulsion. In comparison, micelleplexes of shorter DNA chains precipitated much earlier at higher ionic strength. Interestingly, the chain length influence on the titration diagram seems to be unique to stiff polyanions such as DNA. In comparison, the chain length of flexible polyanions, such as polystyrenesulfonate, shows almost no influence on the titration diagram with cationic micelles.43 This suggests that aggregation of micelleplexes with DNA is primarily due to bridging, while for flexible polyanions, charge neutralization and association of neutralized patches might play more important roles in complex aggregation and therefore show less chain length dependence. Lastly, the length of DNA molecules also influences the equilibration kinetics and long-term stability of the micelleplexes. Previous work has shown that the long-term stability of complexes between cationic micelles and linear polyanions depends on the charge ratio: these complexes showed extended stability in solutions with micelles in excess but precipitated with polyanions in excess.22,43 The long-term stability of micelleplexes observed in this study is consistent with this trend. In addition, the rate of evolution toward thermodynamic structures depends on the DNA length. With DNA in excess, micelleplexes of shorter DNA chains (2442 bp) precipitated within 2 weeks, whereas micelleplexes of longer DNA chains (7537 bp) were soluble for at least 42 days at 20 mM ionic strength. With micelles in excess and at 500 mM, micelleplexes of shorter DNA chains evolved from bimodal size distributions to monomodal distributions within 28 days, while micelleplexes of longer DNA chains showed stable bimodal distributions up to 42 days. The dependence of equilibration kinetics on DNA chain length is not surprising as increasing the length of the polyelectrolytes generally reduces the rearrangement rate.21,29 Unlike DNA length, the topology of DNA showed very limited influence on the properties of the micelleplexes, as shown by turbidimetric titrations, DLS, and cryo-TEM. This might be due to the fact that both the coil size and the persistence length of DNA exceed the hydrodynamic radius of the micelles, and therefore the topology and rigidity differences between linear and supercoiled DNA are less influential. It is also possible that the detailed structures of micelleplexes could differ due to the topology of the DNA on the length scales that were not probed by DLS or cryo-TEM. We do note some subtle differences between micelleplexes of linear and supercoiled DNA. For example, compared to micelleplexes formed with supercoiled DNA, micelleplexes of linear DNA of the same

structure and composition as those of supercoiled DNA, which agrees with the DLS results and confirms that DNA topology only has a minor influence on the structure of micelleplexes.

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DISCUSSION In this study, the influence of DNA length and topology on micelle complexation was explored. This work builds on the existing understanding of micelleplex properties as a function of N/P ratio and solution ionic strength. Overall, this study shows that DNA length has a significant impact on the structure, titration diagram and long-term stability of micelleplexes, while DNA topology has at most a modest influence. DNA length plays an important role in shaping the kinetic structure of micelleplexes. Under kinetic constraints, the DNA chain length dictates the coil size, which in relation to micelle size determines whether the DNA chain can be confined within the corona of one micelle or protrude outward and bridge with other micelles. The structure of micelleplexes therefore depends on the relative size between DNA and the cationic micelle, as revealed by the combination of DLS, SLS, and cryoTEM and schematically summarized in Figure 8. At N/P > 1

Figure 8. Schematic illustrations of micelleplex structures with micelles (N/P > 1) or DNA (N/P < 1) in excess. The micelle and DNA chain sizes on the left are drawn to best reflect their actual hydrodynamic radii. The illustrations employ supercoiled DNA but also apply to micelleplexes of linear DNA. The hydrodynamic radii were measured in acetate buffer at pH 5 and 100 mM ionic strength.

with micelles in excess, micelleplexes can comprise one or multiple micelles that coexist with excess micelles. The size of multiple-micelle complexes increases with DNA length, as longer DNAs are more likely to bridge between multiple micelles. This influence of DNA length is magnified at higher solution ionic strength as electrostatic repulsions are more strongly screened; longer DNA chains can bridge and form large multiple-micelle complexes. In comparison, shorter DNA chains can be fully captured by a smaller number of micelles so that at higher ionic strength, even multiple-micelle complexes are still small. With DNA in excess, increasing DNA length preserves the population of small micelleplexes by providing steric repulsion between complexes. With DNA in excess, micelleplexes can be stabilized by two interactions: electrostatic repulsion from overcharged DNA around the micelle corona and steric repulsions from DNA chains protruding outward from the micelle corona. For shorter DNA, both stabilizing forces are weak such that large aggregates are formed, and which gradually precipitate. For longer DNA, steric repulsions from long DNA chains surrounding micelle corona can stabilize H


Article

Macromolecules

equipment funding from NSF through the UMN MRSEC program under Award DMR-1420013. We appreciate the assistance of Yogesh Dhande with the DNA preparation.

length had slightly larger hydrodynamic radii, which could be due to the fact that linear DNA has a more open conformation that could promote the formation of larger complexes than its supercoiled form. Nevertheless, this reflects one important design principle: the primary structure of the micelleplexes is independent of DNA topology and dependent on the structure of the micelles, the length of the DNA chains, and formation conditions such as solution ionic strength and concentration. As a potential gene delivery vehicle platform, cationic micelles therefore may offer precise control and tunability on the structure and physical properties of their complexes with DNA.

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CONCLUSION The complexation of cationic micelles and DNA produced micelleplexes whose structure and stability were dependent on the length of the DNA molecules, but not their topology. The length of the DNA dictated the probability of one DNA molecule to bridge between micelles as well as the equilibration kinetics of the resultant micelleplexes. Consequently, with micelles in excess, longer DNA chains can bridge between a higher number of micelles and form larger multiple-micelle complexes that lacked evidence of rearrangement, while micelleplexes of shorter DNA formed smaller complexes of similar size as single micelles. With DNA in excess, micelleplexes of shorter DNA formed larger aggregates that eventually precipitated out of the solution while micelleplexes of longer DNA were stable against aggregation. The structure of micelleplexes also affected their instantaneous colloidal stability and led to a shift of inhomogeneous regions to higher N/P ratios with increasing DNA length at high ionic strength. Overall, this suggests that shorter DNA at low ionic strength and low concentration favors formation of small and uniform micelleplexes with micelles in excess.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b02201. Characterization of DNA, turbidimetric titrations, analysis of dynamic and static light scattering data, and representative cryogenic transmission electron microscopy images (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(T.P.L.) E-mail [email protected]. *(T.M.R.) E-mail [email protected]. ORCID

Yaming Jiang: 0000-0002-2623-613X 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 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 I


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Complexation of DNA with Cationic Copolymer Micelles: Effects of

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