Article pubs.acs.org/Macromolecules
Kinetically Arrested Assemblies of Architecturally Distinct Block Copolymers José Luis Santos and Margarita Herrera-Alonso* Department of Materials Science and Engineering, The Johns Hopkins University, Baltimore, Maryland 21218 S Supporting Information *
ABSTRACT: The rapid coassembly of linear and linear−dendritic amphiphiles from homogeneous solution and high supersaturation produces kinetically arrested nanoparticles, the morphologies of which are distinct from equilibrium structures. The binary system of poly(D,L-lactide-co-glycolide)-block-poly(ethylene glycol) with a linear or dendritic architecture of the hydrophilic component, forms spherical hybrid nanoparticles regardless of dendron generation or poly(ethylene glycol) length. Controlled variation in nanoparticle size was achieved through a balance of amphiphile architecture, blend composition, and final solvent content. These results demonstrate how kinetic features of the assembly process influence the formation of hybrid copolymer nanoparticles.
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INTRODUCTION The construction of hybrid systemsthose involving multiple components exhibiting distinct physicochemical characteristicsthrough nondirectional interactions results from a balance between self-organization and integrative coassembly.1 The importance of hybrid systems relies on the fact that their properties can be readily modulated according to composition, affording access to a wide range of materials with distinct physicochemical properties. Mixed surfactant micelles find multiple technological applications for this reason, and extensive experimental and theoretical studies have established the critical molecular attributes underlying mixed micelle formation.2−9 Aggregates from combinations of macromolecular amphiphiles with dissimilar block chemistry have also been discussed.10−13 Aside from the possibilities enabled by the combination of distinct building blocks, the level of complexity of aggregates from macromolecular amphiphiles can also be modulated through kinetic manipulation of the assembly process, yielding structures in different states of equilibrium.14−19 In the case of polymer micelles, the macromolecular nature of the amphiphiles dictates the preferred mechanisms and time scales leading to aggregate relaxation. The slow exchange dynamics observed for highly amphiphilic macromolecular systems, along with controlled variations of block chemistry and/or block sizes has led to the discovery of a multitude of aggregates exhibiting complex geometries.14,19,20 The nonergodic character of these assemblies emphasizes the critical role of kinetic pathways of © 2013 American Chemical Society
polymer assembly on aggregate functional and structural complexity.18,21,22 When assembled from homogeneous solution under conditions of high supersaturation by a rapid change in solvent quality, the nonequilibrium self-assembly of diblock copolymers results in kinetically frozen nanoparticles.15 Aggregate relaxation is precluded when the energetic penalty of solventmediated unimer exchange or particle fusion are high, as would be the case for highly amphiphilic block copolymers. Kinetically frozen nanoparticles are produced by tuning two characteristic time scales: the time to achieve homogeneous mixing and the time for block copolymer self-assembly.23 When this process is carried out in the presence of a hydrophobic solute, hybrid assemblies result. Provided the characteristic aggregation time of the polymer and solute nucleation and growth times are comparable, and homogeneous mixing is ensured, coassembly results in solute-loaded nanoparticles.23 However, if the first of these conditions is not met polymer-stabilized large solute particles or nonloaded (empty) nanoparticles result, depending on which of the two competing processes is faster. Hence, for this method, matching characteristic time scales of multiple simultaneous processes enables the controlled formation of composite systems. This concept has been used to generate drug-loaded nanoparticles.23−27 Received: October 4, 2013 Revised: December 3, 2013 Published: December 17, 2013 137
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It is known that the hydrophobically driven association of molecularly distinct amphiphiles can result in mixed micelles (comicelles), depending on the relative concentration of both species in the medium,6 the comparative molecular characteristics of the blocks comprising the amphiphiles (chemical compatibility and relative chain lengths of both segments),7 and the kinetics of mixed micelle formation.28 However, in the case of aggregates formed by a rapid change in solvent quality, it is unknown if the difference in molecular weights of the amphiphilesand, therefore, differences in characteristic aggregation timeswould impact coassembly and what the resulting morphologies would be. Potential applications of this concept include the formation of protein−polymer nanoparticles.29,30 Herein, we describe the formation of hybrid nanoparticles from architecturally distinct block copolymer amphiphiles by controlling the molar composition of a blend and the kinetic features of the assembly process. Specifically, we examined and contrasted the coassembly of linear and linear−dendritic amphiphiles under equilibrium and nonequilibrium conditions. The latter was achieved by employing a rapid change in solvent quality as the driving force for association. While there are a number of experimental and theoretical studies on the aggregate structures of binary mixtures covering a variety of amphiphilic systems, this is, to the best of our knowledge, the first example that focuses on the combined roles of hydrophilic block architecture and assembly kinetics on hybrid particle formation. We discuss our results in the context of aggregate morphology and particle size.
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Figure 1. (A) Linear (top) and linear−dendritic (bottom) amphiphiles used in this study. (B) Schematic of the coassembly of linear and linear−dendritic amphiphiles into mixed (hybrid) nanoparticles, carried out in a multi-inlet vortex mixer (MIVM) by a rapid change in solvent quality.
PEG was coupled onto hydroxyl dendron ends activated with pnitrophenylchloroformate in high yields. Heterobifunctional PEG chains were terminated with hydroxyl groups (Supporting Information, 9) for most coassembly experiments, lipoic acid (Supporting Information, 10) for gold nanoparticle labeling experiments, and FITC (fluorescein isothiocyanate, Supporting Information, 12) for assessing coassembly through FRET (Förster resonance energy transfer). The hydrophobic block of all amphiphiles was PLGA48 with a comonomer ratio of 1.48 Copolymers were characterized by a combination of NMR spectroscopy and GPC, and were shown to have relatively narrow polydispersity. Nanoparticle assembly was triggered by a rapid change in solvent quality inside a multi-inlet vortex mixer (MIVM Figure 1B). In this process, micromixing occurs in the ms range, affording a homogeneous environment for hydrophobic association.49 Provided the characteristic mixing time is shorter than the self-assembly time of the amphiphile, nanoparticles form with sizes that are independent of mixing velocity.15 In our case, this occurs at Reynolds numbers Re > 2000 (see Supporting Information for calculation of Re based on individual stream velocities). Briefly, a solution of both types of amphiphiles in tetrahydrofuran was rapidly mixed with watera nonsolvent for the PLGA block− at high velocity (Re ∼ 8200 unless otherwise noted) to a final solvent content of 10% (vol). The magnitude and rate of solvent change results in high supersaturation of the amphiphile, the extent of which depends on its critical micelle concentration (Ccmc).15 Ccmc values for the series of block copolymers synthesized were determined by pyrene encapsulation and are given in Table 1. For each family of amphiphiles, Ccmc values increase with wPEG, as expected. Furthermore, Ccmc values were smaller for linear−dendritic amphiphiles with similar HLB to linear ones, as previously reported.45 After mixing, nanoparticle suspensions were dialyzed against water to remove the organic solvent. Particle size and size distributions were measured immediately after the
EXPERIMENTAL SECTION
Details regarding the synthesis and characterization of all amphiphiles studied, as well as processing conditions, are presented as Supporting Information.
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RESULTS AND DISCUSSION The amphiphiles of interest, linear and linear−dendritic copolymers, shared the same chemical identity but differed in the architecture and molecular weight of the hydrophilic block (Figure 1). The linear−dendritic amphiphile was chosen to provide well-defined areas of functional group presentation on a nanoparticle surface. Functional surface “patches” are used to elucidate the synergistic effect between ligand clustering and multivalency for targeted nanoparticles.31−39 Micelles from linear−dendritic amphiphiles are more thermodynamically stable than their linear equivalents and exhibit unique selfassembly behavior.40−46 Linear amphiphiles, on the other hand, are substantially more synthetically tractable and their aggregate kinetics and structures have been exhaustively discussed. To prepare hybrid polymer nanoparticles we synthesized a family of linear and linear−dendritic amphiphiles consisting of biodegradable and biocompatible materials. We targeted the synthesis of amphiphiles with low ( 0.989), while the dashed line is meant as a guide. (C) TEM image and particle size histogram (D) of hybrid nanoparticles from PLGA48-b-PEG14 and PLGA48-b-G2(PEG14) (50% LD component).
philic−lipophilic balance,54 as shown in Figure 3A. HLB values are given in Table 1. Differences in micelle hydrodynamic diameter and corona-forming block size between branched and linear diblocks is attributed to the arrangement of branched blocks in the micellar corona.54,55 Particle core size, also decreased with HLB. While HLB is a useful parameter to compare linear amphiphiles, it fails to capture the molecular complexity of
the linear−dendritic amphiphile. To relate nanoparticle size to the molecular characteristics of the building blocks we have instead used the average mean-square radius of gyration of the hydrophilic block since it explicitly accounts for chain architecture, the parameter that distinguishes the two types of amphiphiles examined. As all copolymers consist of the same hydrophobic block (PLGA 48 ), we focus only on the contribution of the hydrophilic segment. For this, we assume 140
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change in size with concentration (Supporting Information, Figure S7). Studies of polymer micellization have shown that mixed micelle formation is strongly determined by the relative concentration of both species in the medium,6 the comparative molecular characteristics of the blocks comprising the amphiphiles,7 and the kinetics of mixed micelle formation.28 Furthermore, works on binary mixtures of amphiphiles individually forming aggregates of different curvatures have shown the formation of simple and complex micelles and vesicles, and more recently complex multigeometry nanoparticles, depending on amphiphile structure, block incompatibility and blend composition.19,20,46,63,64 Greenall and Gompper have predicted the formation of small vesicles by addition of a lamellar-forming amphiphile to a system exclusively containing a spherical-forming amphiphile, under equilibrium conditions.63 Unlike the morphologies predicted by Greenall, we only observed a bilayer structure for aggregates formed by the self-assembly of PLGA48-b-PEG14; all others appeared to exhibit a spherical morphology. We attribute these deviations to the assembly method since the secondary processes leading to equilibration are energetically unfavorable given low unimer solubility in the continuous phase, steric stability provided by the PEG chains, and slow chain dynamics. Equilibration processes observed in comicellization studies influence aggregate structure, particularly for copolymers with greatly differing lengths of the core-forming block. Esselink et al.28 showed that bimodal distribution of aggregates, composed of pure and mixed micelles, occurred after the initial rapid formation of mixed polymolecular micelles. We followed changes in particle size by DLS over time and observed no difference between freshly dialyzed samples and those aged for approximately four months, suggesting that if rearrangement occurred it was not an interparticle event. Furthermore, we prepared single-component nanoparticles of linear and linear− dendritic amphiphiles, labeled at the hydrophilic ends with FITC and lissamine rhodamine, respectively, to assess the occurrence of unimer exchange or particle fusion/fission by FRET. We did not observe FRET from a blend of these nanoparticles indicating that unimer exchange did not occur within the time scale examined such that mixed assemblies were kinetically stable. The implications of these findings are several. Nanoparticle size, important in a number of fields, can be readily tuned by varying amphiphile composition in a binary mixture. This had been observed before for combinations of linear amphiphiles.50,65 Interestingly though, combinations of structurally distinct amphiphiles allows for a more controlled modulation of particle size compared to the purely linear systems under the conditions examined. The reason for this is unknown but could possibly be due to differences in chain packing for both types of amphiphiles. Furthermore, we have shown that coassembly of different amphiphiles can take place even under conditions that would result in significantly different characteristic individual aggregation times, according to their differences in molecular weight. This effect is similar to the comicellization of block copolymers where one of the components is present at concentrations below its critical micelle concentration but is still incorporated in the composite aggregate.6 It is likely that the presence of the amphiphile with the shortest characteristic aggregation time provides a site for hydrophobic adsorption, so ensuring micromixing takes place is therefore crucial to provide a homogeneous environment for controlled coassembly.
that the dendritic base can be approximated to the junction point of a star polymer for PEG branches (4 or 16 for G2 or G4, respectively). We note that this assumption does not take into account the structural complexity of the dendron and how it may influence chain conformation at the core−corona interface.56 The mean square radius of gyration of the hydrophilic block was estimated considering water as the solvent (correlation length exponent ν = 0.588). For star polymers with f arms, each consisting of N segments,57 ⟨R g 2⟩ ∼ N 2vf 1 − v
where, the number of segments in each arm was calculated based on the number of monomer units in the chain (Nt) and ns the number of monomer units in a Kuhn segment according to
N = Nt /ns for PEO ns = 2.58 Results for all amphiphiles are given in Table 1. As shown in Figure 3, there appears to be a relatively good correlation between aggregate size of spherical nanoparticles and hydrophilic block size (in terms of Rg) for the amphiphiles examined. We would anticipate larger deviations from this trend for the higher generation linear−dendritic amphiphiles in two ways: first, the influence of the dendritic base as the junction point for hydrophilic chains is stronger for the shorter PEG chains and higher generation dendrons (e.g., G4(PEG14)), thus more strongly influencing chain extension;56,59 second, the collapsed PLGA block could prevent tethered PEG chains from adopting all possible conformations due to steric impediments, possibly forcing PEG chains into a more brushlike state.60,61 Co-Assembly via a Rapid Change in Solvent Quality. The characteristic aggregation time of a polymeric amphiphile is determined by its size (a), chain diffusivity (D, also a function of its size), and amphiphile bulk concentration (ϕ)15,62 according to
τ∼
a2 Dϕ2
While the driving force for particle formation is the same for both types of amphiphiles, their characteristic aggregation times may vary considerably. Linear−dendritic amphiphiles have higher molecular weights and lower diffusion constants compared to linear amphiphiles (Table 1), leading to longer aggregation times for a given concentration. According to the experimental conditions used during assembly, characteristic aggregation times of LD amphiphiles can differ by orders of magnitude from linear ones, depending on generation number and PEG chain length. When coassembly was carried out under conditions of rapid mixing and a large solvent jump, aggregates with low polydispersity and controllable sizes were formed, regardless of the combination of amphiphiles used (Figure 4). DLS curves and TEM histograms showed narrow particle size distributions. As shown for PEG14 derivatives (Figure 4B), a constant change in average particle size was achieved by combining LD amphiphiles of different generations and branch lengths; the same was observed for mixed nanoparticles from PEG45. Linear amphiphiles (PLGA48-b-PEG14 and PLGA48-b-PEG45) were also combined at different compositions but, unlike the case of the branched amphiphiles, they did not show a monotonic 141
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Effect of Processing Conditions on Coassembly. The nonergodic character of block copolymer assemblies allows for the development of unique structures resulting from subtle changes in processing conditions.18,66−69 To examine the influence of fabrication method on aggregate structure we studied coassembly under conditions favoring thermodynamic equilibration (i.e., dialysis method). As shown in Figure 5,
for solvent-mediated processes under conditions of high solvent content.15,20,70 Amphiphile architecture did, however, determine the difference in aggregate size among nonequilibrium states. The most pronounced effect of final solvent quality on size was for aggregates of linear amphiphiles, where an increase by a factor of 5 was observed for a 5-fold increase in solvent volume fraction. In contrast, final solvent quality did not greatly affect the sizes of aggregates composed of linear−dendritic amphiphiles: for the same solvent jump, their size only doubled. The binary system of linear and linear−dendritic amphiphiles showed an intermediate behavior. Differences in aggregate size with final solvent content are associated with amphiphile architecture through brush repulsion characteristics and core swelling.15 Chain packing increases with higher final solvent content for both types of amphiphiles, resulting in the formation of larger aggregates. Brush height is expected to increase accordingly. This effect appears to be more pronounced for the bilayer structures formed by the self-assembly of the linear amphiphile. In contrast, the branched nature of the linear−dendritic amphiphile appears to exhibit hindered capacity to aggregation resulting in less efficient interfacial adsorption and thus requiring a larger area along the core−shell interface to relax chain stretching.40−42 The impact of solvent composition on amphiphile packing and aggregate size of mixed nanoparticles is expected to increase with the concentration of linear diblock, as shown in Figure 6B. Additionally, changes in solvent content may also determine the nonequilibrium state at which assembly occurs. Systems with higher unimer solubility will enable solventmediated processes leading to equilibration. This effect may also influence final aggregate size, as shown in Figure 6. Assessment of Coassembly: Labeling Experiments. Particle size analysis suggests a cooperative assembly of linear and linear−dendritic amphiphiles into hybrid aggregates, which was further confirmed by two labeling experiments. In the first, Förster resonance energy transfer (FRET) was used to evaluate amphiphile proximity within a single nanoparticle. The occurrence of FRET is highly dependent on the distance between donor and acceptor, providing a method for the assessment of molecular proximity in the aggregate. If the amphiphiles are close enough (
