Precision Self-Assembly of Amphiphilic Random Copolymers into

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Precision Self-Assembly of Amphiphilic Random Copolymers into Uniform and Self-Sorting Nanocompartments in Water Yuji Hirai,† Takaya Terashima,*,† Mikihito Takenaka,†,‡ and Mitsuo Sawamoto*,† †

Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan RIKEN SPring-8 Center, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan

‡

S Supporting Information *

ABSTRACT: Self-assembly of amphiphilic molecules in water is a cornerstone to build compartmentalized materials toward unique functions, whereas it is yet challenging to create uniform, discrete, and size-controlled nanocompartments. This paper is to report that precision random copolymers, amphiphilic with hydrophilic poly(ethylene glycol) (PEG) and hydrophobic dodecyl pendants, induce precision selfassembly and self-recognition in water to form uniform, tunable, and self-sorting nanoparticles with inner-core hydrophobic compartments covered by PEG chains; the copolymers have been obtained via living or free radical copolymerization. The nanoparticles allow the on-target and predictable control of size, molecular weight, and aggregation number by tuning the primary structure of the copolymers; even mixtures of the copolymers with different composition underwent self-sorting to provide size-controlled discrete compartments.

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INTRODUCTION Compartmentalization via self-assembly and self-sorting plays a vital role to express biological functions in our living systems.1−3 Inspired by nature, various compartmentalized materials (e.g., spherical or rod-like micelles, vesicles) for selective and/or orthogonal functions have been developed via the precision self-assembly of synthetic molecules. On-target self-assembly involves tailor-making sophisticated building blocks including precision synthetic macromolecules (e.g., block copolymers, dendrimers)4−11 and supramolecules,12−15 in turn leading to the precision control of primary structure (molecular weight, composition, monomer sequence), chirality, rigidity, bulkiness, amphiphilicity, and physical interactions, among others. Precision designer molecules, though often comprising complicated structures, also allow self-sorting via self-recognition or self-discrimination, providing discrete complexes and aggregates with different size, composition, and structure in single solutions.2,3,15 However, it is yet challenging to construct uniform or discrete nanocompartments with on-target and predictable size controllability via the self-assembly or self-sorting of accessible compounds. No doubt such self-sorting compartments and systems open new vistas for innovative functions therefrom. Given these backgrounds, we herein report precision selfassembly and self-recognition of amphiphilic random copolymers in water, readily obtained by our metal-catalyzed living radical polymerization or even by conventional free radical processes (Figure 1). Simple in structure and easy to design and synthesize, the precision random copolymers turned out to provide uniform nanoparticles with hydrophobic compartments © XXXX American Chemical Society

inside and covered by hydrophilic polyether pendent chains. The nanoparticle formation is of on-target controllability of size, molecular weight, and aggregation number, while they further afford discrete compartmentalization via hydrophobicity-mediated self-sorting in water. We thus designed amphiphilic random copolymers of poly(ethylene glycol) (PEG) methyl ether methacrylate (PEGMA) and dodecyl methacrylate (DMA)16 via metalmediated living or free radical copolymerization.17,18 The copolymers consist of a hydrophobic polymethacrylate backbone and randomly distributed hydrophilic PEG and hydrophobic dodecyl side chains (Figure 1a). The mass fraction of hydrophilic/hydrophobic units can be easily controlled by monomer feed ratio. In water, the random copolymers selfassemble into micelles carrying hydrophobic cores covered by multiple PEG units. Particularly surprising and unique is that with a proper hydrophilic/hydrophobic balance, these copolymers form, in water, either single-chain or multichain assemblies of a predetermined single overall size or nominal molecular weight that is independent of the degree of polymerization (DP) of the copolymers. In these assemblies of a uniform size, in sharp contrast to amphiphilic block copolymers,4−6 the random copolymers uniquely induce microphase separation of the hydrophilic and hydrophobic pendants along the backbone in water. As a result, apparently, pseudo-Janus structure is formed Received: May 21, 2016 Revised: June 27, 2016

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DOI: 10.1021/acs.macromol.6b01085 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

Figure 1. Self-assembly of amphiphilic random copolymers in water. (A) Design of amphiphilic random copolymers with different degree of polymerization (DP) and hydrophobicity (P1−P33) via living or free radical copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and dodecyl methacrylate (DMA). FRP: free radical polymerization. (B) PEGMA/DMA random copolymers induce intermolecular and/ or intramolecular self-assembly to form uniform nanoparticles in water. Multiple hydrophilic PEG chains and hydrophilic dodecyl units are phaseseparated along the polymethacrylate backbone, locally forming pseudo-Janus-type structure at the particle interface. (C) Effects of DP and DMA composition on the intermolecular or intramolecular self-assembly of the random copolymers in water. The copolymers have clear threshold DP (red dashed line) between intermolecular (dark blue circle) and intramolecular (open circle) self-assembly; the threshold DP increased with increasing DMA composition.

toluene at 80 °C (for experimental details, see the Supporting Information). DP and DMA composition of the polymer samples were illustrated in Figures 1A and C. As verified by size-exclusion chromatography (SEC) and proton nuclear magnetic resonance (1H NMR), all copolymers were well controlled with narrow molecular weight distribution and ontarget composition (m/n) (Mn = 10 800−133 000, Mw/Mn = 1.2−1.4, by PMMA calibration, Figures S1−S5, Tables S1 and S2). As typically shown in Figure 2A (black line, by PEO calibration), the SEC curves of the copolymers with 40 mol % DMA in N,N-dimethylformamide (DMF) clearly shifted to high molecular weight with increasing DP. Determined by multiangle laser light scattering coupled with SEC (SECMALLS) in DMF, absolute weight-average molecular weight [Mw,DMF (MALLS)] of the products also linearly increased with DP [Mw,DMF (MALLS) = 20 800−91 400 g/mol, Table S1]. In all cases, Mw,DMF (MALLS) was fully consistent with the molecular weight calculated from corresponding numberaverage molecular weight by 1H NMR and Mw/Mn [Mw,DMF (MALLS) ∼ Mn (NMR) × Mw/Mn]. In addition, PEGMA/DMA random copolymers with broad molecular weight distribution (P18: DMA = 40 mol %, Mn = 19 300, Mw/Mn = 2.3; P33: DMA = 50 mol %, Mn = 18 200, Mw/Mn = 2.4) were also prepared via free radical copolymerization of PEGMA and DMA with 2,2′-azodiisobutyronitrile. Self-Assembly of PEGMA/DMA Random Copolymers in Water. The self-assembly of PEGMA/DMA copolymers (P1−P33) in water was investigated in detail with SEC, MALLS, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and 1H NMR. In sharp contrast to the “as-

at the compartment interface to effectively stabilize the hydrophobic cores with outer PEG chains. Amphiphilic and/ or functional random copolymers often undergo “intramolecular” self-assembly into unimer micelles16,19,20 and single-chain polymeric nanoparticles.21−29 However, beyond such general understanding and consequence, we revealed clear regularity and innovative controllability for “intermolecular” self-assembly in water: (1) PEGMA/DMA random copolymers have a clear threshold DP between intermolecular and intramolecular self-assembly; the threshold DP increased with the hydrophobicity (Figure 1a). (2) The copolymers below a threshold DP intermolecularly self-assemble into uniform nanoparticles with constant size and molecular weight; these factors are controllable just with DMA composition. (3) The aggregation number of polymers in nanoparticles can be predictably controlled in small numbers (e.g., 2, 3, 4, 5, ..., 12) by DP and/or DMA composition. (4) The nanoparticles are thermodynamically stable in a wide range of concentration and for a long-term (e.g., 0.02−100 mg/mL; for over 4 months). (5) Self-sorting of the copolymers depends simply and exclusively on the hydrophobicity (DMA composition) to form discrete nanoparticles with different hydrophobicity and size.

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RESULTS AND DISCUSSION Synthesis of PEGMA/DMA Random Copolymers. A series of PEGMA/DMA random copolymers (P1−P17, P19− P32) with different chain length (DP = 40−630) and hydrophobicity (DMA: 20, 30, 40, 45, 48, and 50 mol %) were synthesized by Ru(Ind)Cl(PPh3)2/n-Bu3N-mediated copolymerization of PEGMA (Mn = 475, an average oxyethylene unit of 8.5) and DMA with a chloride initiator in B

DOI: 10.1021/acs.macromol.6b01085 Macromolecules XXXX, XXX, XXX−XXX

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Figure 2. Precision intermolecular self-assembly of PEGMA/DMA random copolymers into uniform nanoparticles in water. (A) SEC curves (by refractive index detector) of the 40 mol % DMA copolymers with different DP (44: P10, 52: P11, 69: P12, 102: P14, 190: P16, 424: P17) by living radical polymerization and that (P18) by free radical polymerization (FRP) in DMF (black lines) and H2O (blue lines). The apparent molecular weight for P10−P16 was almost constant in H2O (peak-top molecular weight: Mp = ∼ 14 000) though the molecular weight linearly increased with DP in DMF, indicating that they induce inter- or intramolecular self-assembly in water to form uniform nanoparticles with identical size. P18 with broad molecular weight distribution simultaneously undergo inter- or intramolecular self-assembly in water. (B, C) Absolute weight-average molecular weight [Mw (MALLS)] of 40 mol % DMA copolymers (B: P10−P16) and 50 mol % DMA counterparts (C: P25−P31) in DMF (black circle) and H2O (blue circle). The copolymers formed uniform nanoaggregates with almost constant Mw (MALLS) in H2O, dependent on DMA content (40 mol %: Mw,const = ∼100 000, 50 mol %: Mw,const = 220 000). Aggregation number in water: Nagg = Mw,H2O (MALLS)/Mw,DMF (MALLS).

expected” results in DMF, strikingly different behavior has been observed for the same set of copolymers dissolved in water. First, copolymers with 40 mol % DMA (P10−P17) were analyzed by SEC and MALLS in water. Here, to compare the apparent size in water or DMF, PEO calibration was used for SEC measurements in the both solvents. P10−P16 with DP = 190 or below exhibited unimodal and narrow SEC curves virtually identical in regard to shape, narrowness, and peak-top molecular weight (Mp = ∼ 14 000 g/mol; Mw/Mn = 1.2, Figure 2A, blue lines). For all the samples, the absolute weight-average molecular weight determined by MALLS detector [Mw,H2O (MALLS)] was also constant and approximately 100 000 g/ mol (Figure 2B). The constant Mw,H2O (MALLS) corresponded to that for a single-chain folding P16 (DP = 190), but for the samples of smaller DPs, the value implied multichain assemblies, the aggregation number of which depended on

DP. Importantly, these results show that the copolymers selfassembled in water to form nanoaggregates with identical size and nominal molecular weight, independent of the DP. As a result, the aggregation number of P10−P16 in water [Nagg = Mw,H2O (MALLS)/Mw,DMF (MALLS)] can be predictably controlled: Nagg = 1 (single-chain folding, P16: DP = 190), 2 (P14: DP = 102), 3 (P12: DP = 68), 4 (P11: DP = 52), and 5 (P10: DP = 44) (Figure 2B). Nagg thus increased with decreasing DP. The radius and structure of the nanoaggregates (40 mol % DMA) in water were further analyzed by DLS and SAXS. The hydrodynamic radius (Rh; by DLS) of the copolymers in water was also almost constant, whereas those in DMF gradually increased with DP ([polymer] = 10 mg/mL, Rh = ∼ 5.0 nm in H2O, 4.1−7.0 nm in DMF, Figure 3A and Table S3). SAXS analysis with Guinier plot showed that in water copolymers C

DOI: 10.1021/acs.macromol.6b01085 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules with different DP (78: P13, 102: P14, 190: P16) all formed spherical nanoparticles with an almost same radius of gyration (Rg = 3.2−3.5 nm) (Figure 3B and Table S3). These highly regulated self-assemblies are triggered by hydrophobic interaction of the polymethacrylate backbone and dodecyl pendants, as confirmed by 1H NMR spectroscopy in D2O where the proton signals turned broader (Figure S10). These results demonstrate that the amphiphilic random copolymers (40 mol % DMA) of DP below about 200

Figure 4. Precision control of aggregation number [Nagg = Mw,H2O (MALLS)/Mw,DMF (MALLS)] of copolymers in water by DP and DMA composition.

S2), while the aggregation number increased accordingly (Figure 4). It should be noted that the aggregation number in water is thereby precisely and predictably controlled in small numbers (e.g., 2, 3, 4, 5, ..., 12) by changing DP and composition (Figure 4): Nagg is inversely proportional to DP in the case below threshold DP. This predictable controllability of aggregation numbers would be quite effective for the precision functionalization of nanocompartments. Additionally, all the copolymers had threshold DP between intermolecular self-assembly and intramolecular counterpart; the DP increased with increasing hydrophobic DMA content (Figure 1A). Another rather surprising observation is that a narrow molecular weight distribution (MWD) is apparently not a critical factor for the uniquely controlled self-assembly in water. Namely, similar aggregation did occur for copolymers with a broad MWD (P18, P33: Mw/Mn = 2.3−2.4, DMA = 40 or 50 mol %), prepared by free radical copolymerization. In sharp contrast to their broad SEC profiles in DMF, these copolymers gave narrow and unimodal distributions in water (Mw/Mn = 1.2−1.3), strikingly akin, in both shape and position (Mp), to those for the corresponding controlled copolymers with narrow MWDs by living radical polymerizations (Figure 2A and Figure S7). Here, smaller (lower DP) chains intermolecularly selfassembled into multichain aggregates, while larger (higher DP) chains intramolecularly self-folded, both heading to uniformsize nanoparticles of a virtually single molecular weight. Thus, the copolymers in water form most thermodynamically stable aggregates, either unimolecular or multimolecular depending on component chain size. The self-assembly of PEGMA/DMA random copolymers via free radical polymerization is likely one of the readily accessible systems to obtain size-controlled uniform nanocompartments because “living” polymerization or cumbersome fractionation is no longer required therein. Origin of Precision/Predictable Self-Assembly. To clarify the origin of controlled and predictable self-assembly, we examined a series of copolymers of varying DMA contents and DP, each of which gives a constant molecular weight of nanoparticles (Mw,const): DMA content = 20, 30, 40, 45, 48, and 50 mol %; Mw,const = 33 000, 53 000, 100 000, 130 000, 180 000, and 220 000 g/mol, respectively (Figure 5A and Figure S9). A particular Mw,const here corresponds to the threshold molecular weight for a single-chain folding polymer with a respective DMA composition. As seen in the logarithmic plots in Figure 5D, Mw,const is proportional to the power of three of hydrodynamic radius Rh by DLS. This importantly demonstrates that all of the nanoaggregates have a spherical structure

Figure 3. (A) DLS intensity distribution and (B) SAXS profiles of 40 mol % DMA copolymers (P13: DP = 78, P14: DP = 102, P16: DP = 190) in water and P13 in DMF at 25 °C ([polymer] = 10 mg/mL).

intermolecularly self-assemble in water to form spherical uniform nanoparticles with identical molecular weight, size, and density. In contrast, larger copolymers (P16 and P17; DP > 200) underwent intramolecular single-chain folding, where the apparent molecular weight increased with DP. Thus, PEGMA/DMA amphiphilic random copolymers with 40 mol % DMA clearly have a threshold DP (∼200) between intermolecular self-assembly and intramolecular counterpart (single-chain folding) (Figure 1C). These uniform nanoparticles are thermodynamically stable and retained their size and aggregation states for a long time (>4 months) in a wide range of concentrations (0.02−100 mg/ mL, Rh =