Article Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Packaging pDNA by Polymeric ABC Micelles Simultaneously Achieves Colloidal Stability and Structural Control Yaming Jiang,† Timothy P. Lodge,*,†,‡ and Theresa M. Reineke*,‡ †
Department of Chemical Engineering & Materials Science, University of Minnesota, 421 Washington Avenue SE, Minneapolis, Minnesota 55455, United States ‡ Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States
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ABSTRACT: Compaction of DNA by oppositely charged nanoparticles is a fundamental phenomenon in nature and of great interest to developing therapeutics. In addition, the ability to orthogonally control the composition and structure of interpolyelectrolyte complexes is needed to develop materials for diverse applications. Herein, we systematically investigate the complexation of plasmid DNA and polymeric cationic AB and ABC micelles to explore the influence of micelle outer nonionic corona length on the colloidal stability, size, composition, and structure of the resulting “micelleplexes”. The micelles were selfassembled from amphiphilic block polymers, poly(ethylene glycol)-block-poly((2-dimethylamino)ethyl methacrylate)-blockpoly(n-butyl methacrylate) (PEG-b-PDMAEMA-b-PnBMA), and PDMAEMA-b-PnBMA with the same Mn of PDMAEMA. These spherical micelles have similar hydrodynamic radii and core sizes, but the Mn of the outer PEG block ranged from 0 to 10 kDa. The colloidal stability of micelleplexes as a function of stoichiometric charge ratio was assessed by turbidimetric titration and was found to dramatically improve with the addition of an outer PEG corona, even as short as 2 kDa. With the use of a combination of dynamic and static light scattering, ζ-potential, and cryogenic transmission electron microscopy, it was found that the size, composition, and structure of micelleplexes are closely correlated with the Mn of the PEG block. Indeed, these micelleplexes were found to adopt beads-on-a-string morphologies that resemble the general structure of chromatin, and the number of micelles per micelleplex systematically decreased with increasing PEG length. These findings demonstrate the power of polycationic micelles to condense DNA into biomimetic structures and provide a mechanistic understanding of nucleic acid complexation and of how micelle architecture affects the properties of micelleplexes, while offering an appealing strategy to control the properties of micelleplexes by tuning a single parameter.
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tertiary structures,19−21 and their PECs need to have superb colloidal stability in complex media and adopt specific shapes or sizes to retain and enable biological functions.22−24 However, robust control over these PECs remains challenging. In contrast, biological systems routinely exploit and control hierarchical PECs; for example, complexation of DNA with histone octamers controls compaction of chromatin by several thousand-fold within the nucleus of all cells.25−27 The stability and lability of these DNA-histone complexes are also intimately related to the regulation of gene expression,28 to understanding fundamental processes such as embryonic development28 and regulation of metabolism,29 and for development of cancer therapeutics.30 Therefore, establishing the correlation between polyion architecture and chemistry with the structure and stability of the resulting PECs is important not only for developing and optimizing polyions for
INTRODUCTION Complexation between oppositely charged polyelectrolytes is of fundamental importance to understanding and developing versatile and responsive materials for a wide range of applications, such as filtration,1 surface modification,2 and therapeutic delivery.3,4 Polyelectrolyte complexes (PECs)5,6 are inherently responsive to solution ionic strength,7,8 and their solubility depends heavily on the stoichiometric charge ratio8,9 and the chemical identity10 of the composing polyions, which can be readily tuned via modern synthetic techniques.3,4,11 The structure of PECs is sensitive to the architecture of the composing polyions, particularly when the polyions are nanoparticles, such as brushes,12 or self-assembled structures such as micelles.13−15 The structure and colloidal stability of PECs can potentially be controlled through polyion design, and such control is essential for many applications. For example, many polyions are designed to interact with charged biomacromolecules for biomedical applications, such as protein encapsulation16 and RNA/DNA delivery.3,17,18 These charged biomacromolecules adopt complex secondary and © XXXX American Chemical Society
Received: June 14, 2018
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DOI: 10.1021/jacs.8b06309 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Article
Journal of the American Chemical Society
solution environment. The detailed mechanism of DNA compaction within a micelleplex is also less understood. Few of these micelleplexes observed exhibit the structural regularity observed in chromatin, even though theory and simulations on complexation between DNA and cationic spheres have predicted that DNA can wrap around cationic spheres in regular solenoid patterns.54,55 Understanding the relation between polycation structure and micelleplexes would allow both fundamental understanding of DNA condensation in biological systems and applied control for packaging nucleic acids for therapeutic applications. Herein, we seek to understand how polycationic micelle corona architecture affects the colloidal stability, composition, and structure of their complexes with pDNA. Our previous work has shown that the stiffness of pDNA promotes bridging of core−shell cationic micelles, leading to poor colloidal stability when DNA is in excess.46 We hypothesize that the addition of a hydrophilic nonionic block as the micelle outer corona can effectively improve the colloidal stability of micelleplexes over a wide range of stoichiometric charge ratios and modulate the bridging mechanism of DNA chains among micelles to tune the size and composition of the subsequent micelleplexes. To test this hypothesis, we systematically investigate the complexation between pDNA and spherical cationic micelles of core−shell and core−shell−corona structures, as shown in Scheme 1. Here, the hydrophilic
diverse applications but also for elucidating fundamental biological processes. The unique features of DNA result in interesting solution structures. Double-stranded DNA adopts a helical structure that is semiflexible with a persistence length of 50 nm,31,32 is highly charged (the B-form has an axial charge density of 6 e−/ nm), and can exhibit long contour lengths for plasmids (∼ to >1 μm). In contrast to PECs with flexible polyions, complexes of DNA with polycations can exhibit unique and unanticipated structures; for example, complexes with multivalent cations and polylysine homopolymers have been found to form rodlike and toroidal structures.33,34 However, complexes of DNA and cationic homopolymers often suffer poor colloidal stability and are stable only under selective conditions.35,36 Diblock polymers with a hydrophilic nonionic block and a cationic block complex with plasmid DNA (pDNA) and usually form stable “polyplexes”, in which neutralized pDNA condenses and forms a core that is solubilized by hydrophilic blocks of the polymers.17,18,37−39 For example, Takeda et al. have shown that the shape of these core−shell polyplexes can range from globular to rodlike depending on the relative lengths of the hydrophilic and cationic blocks.38 Cationic dendrimers have also been extensively studied and found to complex doublestranded DNA into “dendriplexes”,40−43 and Ainalem et al. have shown that these dendriplexes can adopt globular, cylindrical, or toroidal shapes depending on the dendrimer generation when negative charge from the DNA is in excess; dendriplexes with higher generation dendrimers are more prone to aggregation.41 In such cases, subtle variations in the polycation architecture substantially influence the resulting shapes and colloidal stability of DNA complexes. Polycationic micelles are related systems that consist of selfassembled amphiphilic cationic polymers, and they can complex with DNA to form “micelleplexes”.44−47 The structure of these micelles can be readily tuned48−50 to either mimic a histone octamer, or to adopt a wide range of shapes, such as spheres,49 disks,51 and cylinders,50,52 where structural parameters can be systematically varied. In addition, the extremely slow chain-exchange kinetics of micelles with long hydrophobic core blocks ensures that micelle shape is invariant during complexation.53 Further, the components of a micelle corona can be varied from purely cationic to a mixture of cationic and nonionic components in statistical, gradient, or block distributions, which have been shown to influence the structure, rearrangement kinetics, and colloidal stability of their complexes with oppositely charged flexible polyelectrolytes.13,14 Previous studies on micelleplexes with spherical micelles in excess show that the pDNA chains bridge multiple micelles to form large aggregates.44−47 For core−shell micelles with a pure cationic corona, Rinkenauer et al.45 and Jiang et al.47 have observed micelleplexes composed of multiple micelles linked by DNA chains. For micelles with a mixed corona of cationic and hydrophilic nonionic chains, Sharma et al. observed that plasmids condense into rodlike structures with micelles decorating the surface.44 With cationic core− shell micelles without a hydrophilic nonionic outer layer, these micelleplexes were unstable in solution and precipitated with excess pDNA.46,47 Despite these recent studies, many factors affecting the properties of the micelleplexes remain unexplored. For example, little is known about how the architecture and corona chemistry of the micelles affect the structure and colloidal stability of the resulting micelleplexes or how the size of the micelleplexes can be controlled independently of
Scheme 1. Cationic Micelles Self-Assembled from Amphiphilic Block Polymers and Their Micelleplexes with pDNA
nonionic block length systematically increases while the cationic block length and the micelle core radius remain constant. The colloidal stability of micelleplexes was assessed by turbidimetric titration, and micelleplex structures were characterized by a combination of dynamic and static light scattering and cryogenic transmission electron microscopy. In general, it was discovered that micelleplexes with a hydrophilic outer corona exhibit colloidal stability over a much wider range of charge ratios. With micelles in excess, the micelleplexes adopt beads-on-a-string structures and the nonionic corona length controls the number of micelles per pDNA. In particular, longer PEG chains converge onto micelleplex structures with on average two micelles and two DNA chains per micelleplex. This study is the first to show that cationic micelles with distinct core−shell−corona structure with a hydrophilic nonionic outer layer have the remarkable ability to maintain superior colloidal stability of the micelleplexes over a wide range of formations, including high salt concentrations. In addition, such core−shell−corona micelles can compact B
DOI: 10.1021/jacs.8b06309 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Article
Journal of the American Chemical Society
dynamic light scattering (DLS) shows that the mean hydrodynamic radii (Rh) of these micelles are around 30 nm, with narrow dispersities as shown in Figure 1. Small-angle Xray scattering (SAXS) data for dilute ODB micelles were collected, and due to the low contrast (in Figures S12−S15), only Rc was extracted as ∼12 nm, as shown in Table 1. Due to core block hydrophobicity and high Mn, these micelles were kinetically trapped with respect to chain exchange.53 The micelle composition was characterized by static light scattering (SLS), which gives the weight-averaged molar mass (Mw), as shown in Table 1. Assuming the cores were free of solvent and had the same density as the core block homopolymer, the micelle core sizes were calculated and found to agree very well with Rc values measured by SAXS. The Rc decreases slightly with increasing O block length. As calculated from the micelle Mw, the aggregation number increases with core block length but decreases with increasing corona length, as expected.65 The number of amine groups per micelle was also calculated. For ODB micelles, the number of amine groups decreases with increasing Mn,O, while the DB micelles have the fewest amine groups, (16 ± 2) × 103. In comparison, the pDNA carries only 4884 phosphate groups per chain; at equal numbers of ODB(10) micelles and pDNA chains, the +/− charge ratio is 5. On the other hand, pDNA chains are extended coils, as the persistence length is ∼50 nm.31,32 They have Rh ≈ 42 nm in acetate buffer of pH 5 and 500 mM ionic strength47 and are therefore larger than the micelles when free in solution. Furthermore, the contour length of pDNA is 830 nm, which is more than an order of magnitude larger than the micelle Rh. The significant disparity between pDNA and micelle structure therefore poses an interesting question: can a semiflexible pDNA chain condense around the corona of a single micelle, which is both more highly charged and more compact? Enhancement of Micelleplex Colloidal Stability. Micelleplex formation and solubility were explored using turbidimetric titration as a function of +/− charge ratio. The pDNA (or micelle) solution was gradually titrated into the micelle (or pDNA) solution, while the complexation process was monitored by solution transmittance as a function of +/− charge ratio. Since the total mass concentration of the titration solution was maintained constant, a decrease in solution transmittance as the +/− charge ratio approaches 1 reflects an increase in the average particle size. Precipitation was soon observed whenever the transmittance fell below 0.9 (Figures S16−S18). The results reveal that the addition of the PEG outer corona greatly improves the colloidal stability of micelleplexes, over a significantly wider range of charge ratios. Figure 2 summarizes the solubility of micelleplexes as a function of solution ionic strength and Mn,O. The highlighted inhomogeneous regions show the range of +/− charge ratios over which micelleplexes precipitate at a given ionic strength. As Figure 2a shows, the inhomogeneous region of DB micelleplexes is confined to +/−
