Synthesis and Nano-object Assembly of Biomimetic Block Copolymers

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Synthesis and Nano-object Assembly of Biomimetic Block Copolymers for Catalytic Silver Nanoparticles Shinji Sugihara, Masahiro Sudo, and Yasushi Maeda Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01558 • Publication Date (Web): 15 Aug 2018 Downloaded from http://pubs.acs.org on August 17, 2018

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Synthesis and Nano-object Assembly of Biomimetic Block Copolymers for Catalytic Silver Nanoparticles

Shinji Sugihara*, Masahiro Sudo, and Yasushi Maeda

Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan

*Correspondence to Shinji Sugihara (E-mail: [email protected])

ABSTRACT Biomimetic

ABC

triblock

copolymers

phosphorylcholine]-b-poly[2-(dimethylamino)ethyl

of

poly[2-(methacryloyloxy)ethyl

methacrylate]-b-poly(2-hydroxypropyl

methacrylate) (PMPC-b-PDMA-b-PHPMA) were synthesized by RAFT aqueous dispersion polymerization of 2-hydroxypropyl methacrylate (HPMA) in the presence of a PMPC-b-PDMA macromolecular chain transfer agent (macro-CTA). This ABC triblock copolymer deploys well-known biocompatible PMPC and PDMA for the coordination of Ag+ ions to form silver nanoparticles in situ on reduction, and PHPMA for assembling (core) in water. The synthesis of PMPC-b-PDMA-b-PHPMA starts when both the reactive steric stabilizer of PMPC25-b-PDMA4 macro-CTA and HPMA monomer are dissolved in water. The growing PHPMA is not soluble in water and begins to assemble based on three-layer onion micelles, in which the outer and inner

shells

are

PMPC

and

PDMA,

respectively.

In

the

synthesis

of

PMPC25-b-PDMA4-b-PHPMAz at a constant 25 w/w% solids concentration, the resultant assemblies change from spheres to worms to jellyfishes to vesicles when the targeted PHPMA chain length increases from 100 to 400mer at full monomer conversion. Furthermore, in the synthesis of identical PMPC25-b-PDMA4-b-PHPMA400 copolymers, the assembly morphology can be controlled from vesicles to spheres through worms by varying the solids concentration in the polymerization mixture, decreasing from 25 to 15 w/w% at full monomer conversion. Thus, the final morphology can be tuned by the degree of polymerization of HPMA and the solids concentration

in

the

polymerization

mixture.

Using

the

resultant

three

PMPC25-b-PDMA4-b-PHPMA400 assemblies as scaffolds, Ag(0) nanoparticles (Ag-NPs) are

ACS Paragon Plus Environment

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obtained through in situ reduction of AgNO3 facilitated by electrostatic interactions between the Ag+ ions and PDMA moieties. The resultant Ag-NPs loaded in the assemblies exhibit excellent stability, dispersibility and activity of catalyst for the reduction of p-nitrophenol. The order of rate constants for the reduction using Ag-NPs loaded in the assemblies is worms > vesicles > spheres, which corresponds to the order of the surface areas of the assemblies of PMPC25-b-PDMA4-b-PHPMA400. These results can be achieved thanks to the kinetically-frozen PMPC25-b-PDMA4-b-PHPMA400 assemblies with identical compositions.

Introduction The self-assembly of block copolymers to form various macromolecular nanostructures is known in both solid and liquid phases, with various prominent functions stemming from the structure. In particular, amphiphilic diblock copolymers have been demonstrated to form characteristic aggregates in selective solvents. The fundamental driving force for solution self-assembly is the solvophobic effect (hydrophobic effect in aqueous solution). This is well-documented in various reviews.1-5 For an amphiphilic diblock copolymer in a selective solvent for one segment, the precise nanostructure, i.e., assembly morphology, is primarily determinable by the inherent molecular curvature. The curvature is related to the surfactant packing parameter, P. The parameter depends on the relative core-block volume (v) divided by the product of the effective interfacial area (a0) at the core-shell/solvent interface and the chain length normal to the surface per molecule (l0):

P=

v a0l0

(1)

Spherical micelles are produced when P ≤ 1/3, cylindrical (rod-like) micelles are favored when 1/3 < P ≤ 1/2, and vesicles are formed when 1/2 < P ≤ 1. Although vesicles are flexible bilayer aggregates, the planar bilayer of lamellae is ideally produced when P = 1. This concept was originally introduced by Israelachvili and co-workers6,7 to explain the self-assembly of small molecule surfactants, and then was applied to a diblock copolymer self-assembly system by Antonietti and Förster.8 Therefore, if the solubility between the blocks differs greatly for a particular solvent, the AB diblock copolymer morphology can generally be dictated by the ratio of the segment length in shell to that in the core at a constant polymer concentration. In many cases, when synthesizing such an AB block copolymer nanostructure in solution, the A block and then the B block are sequentially synthesized in a good solvent by living/controlled polymerization. In the case of the radical polymerizations, this includes atom transfer radical polymerization

(ATRP),9-12

nitroxide-mediated

polymerization


(NMP),13,14

iodine

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transfer polymerization (ITP),15,16 organotellurium-mediated radical polymerization (TERP), 17,18 cobalt-mediated radical polymerization (CMRP),19 reversible addition-fragmentation chain transfer (RAFT) polymerization,20-22 and reversible chain transfer catalyzed polymerization.23,24 The resulting polymer is then purified by an appropriate method and organized in a selective solvent. However, this may include complicated processing steps. On the other hand, there has been considerable interest in synthetic approaches leading to the formation of in situ block copolymer self-assembles. This approach

is well-known as polymerization-induced

self-assembly (PISA), which requires fewer processing steps in its implementation. One PISA formulation, RAFT dispersion polymerization, is known to be a much simpler formulation for self-assembly since the initial reaction mixture is homogeneous and the block copolymer architecture, which can be controlled by the abovementioned packing parameter, P, can be directly constructed. For such formulations, special monomers are necessary; the monomer dissolves in water and the polymer does not dissolve in water. Often this will cause macroscopic precipitation. However, when using a reactive steric stabilizer, stable colloidal dispersions can be obtained.25 Although many examples have been reported for PISA formulations, there are well-documented reviews by Armes,26-28 Pan,29 Charleux,30 and Lowe.31 In 2011, Sugihara and Armes et al. reported the first production of “biocompatible” poly[2-(methacryloyloxy)ethyl phosphorylcholine] (PMPC)-based spheres, worms and vesicles with a methacrylate core using a PMPC macromolecular chain transfer agent (macro-CTA) as a reactive steric stabilizer via PISA formulation of an aqueous dispersion polymerization.32 The phosphorylcholine motif including biocompatible PMPC is a significant component of cell membranes. Such phosphorylcholine-based polymers can be used to produce surfaces that are outstandingly resistant to bacterial/cellular adhesion and protein adsorption, thereby many biomedical applications have been developed.33-35 This is thanks to the very unique hydration state of PMPC, whose MPC repeat unit is known to be associated with many waters (up to 24 molecules).36,37 Thus, PMPC is polymerization.

32,38-40

an excellent steric stabilizer for aqueous dispersion

For example, by using 2-hydroxypropyl methacrylate (HPMA) as the core

monomer for the chain extension polymerization in water for PMPC macro-CTA, PMPC-b-PHPMA nanoparticles with various morphologies were synthesized directly in concentrated aqueous solution. Significantly, each morphology could be targeted by simply changing the final solids concentration (the weight percentage of formed PMPC-b-PHPMA in water). For many macromolecular amphiphiles, in contrast to small-molecule amphiphiles with equilibrium morphology, the rate of unimer exchange between assemblies and individual diblock copolymer chains is negligible, leading to a range of kinetically frozen, i.e., nonergodic, structures at room temperature.4 However, thanks to this formulation for PMPC-b-PHPMA nanoparticles via RAFT aqueous dispersion polymerization, the formidable technical challenge


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of constructing different morphologies using identical and kinetically-frozen block copolymers has been overcome. Since different morphologies can be obtained from exactly the same block copolymer (i.e., both the composition ratio and structure are the same) using aqueous dispersion polymerization,32,41 morphological applications such as templates for inorganic nanoparticles and biomedical applications have attracted broad scientific interest. Therefore, we explored the function of PMPC-b-PHPMA as a biomimetic template for inorganic compounds. However, in preliminary experiments, PMPC-b-PHPMA did not function well as a template for Ag+ ions and reduced Ag(0) nanoparticles (Ag-NPs). Therefore, novel biomimetic ABC triblock copolymer PMPC-b-PDMA-b-PHPMA was synthesized by the route shown in Figure. 1 on the basis of PMPC-b-PHPMA.32 The poly[2-(dimethylamino)ethyl methacrylate] (PDMA) segment easily bonded with inorganic ions such as Ag+ and Au3+ to act as an effective stabilizing and reducing agent in nanoparticle manufacturing, possessing bactericide properties and biocompatibility.42-44 Thus, PMPC-b-PHPMA including the PDMA segment would be available as a scaffold for Ag-NPs prepared by in situ chemical reduction, and the resulting Ag-NPs loaded PMPC-b-PDMA-b-PHPMA assemblies could be applied to biomimetic catalysis. However, as far as we are aware, there have been no reports on biomimetic PMPC-based Ag-NPs catalysts. In addition, there are only a few inorganic/polymer composites using block copolymer assemblies with variable morphologies via PISA formulation, such as Au,45,46 Ag,47-49 SiO2,50 iron oxide,51 and zinc tetraphenylporphyrin.52 Among these, there are no examples comparing the catalytic action using kinetically-frozen block copolymer assemblies of exactly the same composition and polymer structure but different morphologies. Herein, we report the RAFT aqueous dispersion polymerizations of HPMA using PMPC-b-PDMA macro-CTA (PMPC25-b-PDMA4 macro-CTA) at 70 ºC wherein the PMPC-b-PDMA

macro-CTA

is

extended

to

produce

kinetically-frozen

PMPC-b-PDMA-b-PHPMA triblock copolymer assemblies in water (Figure 1). The polymerization under the various solids concentrations allows the formation of various morphologies such as spheres, worms, jellyfishes, and vesicles even from the exact same PMPC24-b-PDMA4-b-PHPMA400. Furthermore, when AgNO3 is introduced into these assemblies, silvered PMPC24-b-PDMA4-b-PHPMA400 assemblies with the same Ag contents are formed in situ. The resulting Ag-NPs are directly observed in PMPC-b-PDMA-b-PHPMA assemblies without staining by transmission electron microscopy (TEM). These Ag-NPs loaded assemblies are considered to be biomimetic catalysts, because they exhibit good colloidal stability owing to the biocompatible PMPC outer shell. The assemblies possess catalytic activity depending on the surface area of each morphology for the reduction of p-nitrophenol (p-NP) with NaBH4.


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Figure 1. (a) Synthesis of PMPC (PMPC25) and (b) PMPC-b-PDMA (PMPC25-b-PDMA4) macro-CTAs via RAFT solution polymerization, and (c) RAFT aqueous dispersion polymerization of HPMA using this PMPC25-b-PDMA4 macro-CTA at 70 ºC. Using this facile approach, spheres, worms, jellyfishes, and vesicles can be directly prepared, depending on either the total solids concentration or the mean degree of polymerization of the PHPMA block.

Experimental Section


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Materials. 2-(Methacryloyloxy)ethyl phosphorylcholine (MPC, Sigma-Aldrich; >97.0% or partly

donated

from

NOF

corporation)

were

used

without

further

purification.

2-(Dimethylamino)ethyl methacrylate (DMA, TCI; >98.5%) and HPMA (TCI; > 97.0 % with the isomeric mixture53) were purified using inhibitor removers prepacked columns (Sigma-Aldrich). 4,4’-Azobis(4-cyanopentanoic acid) (V-501, Sigma-Aldrich) was purified by recrystallization from methanol. 4-Cyanopentanoic acid dithiobenzoate (CADB) was synthesized according to a literature protocol.54 p-NP (TCI; >99.0%) was used without further purification. Synthesis of PMPC, PMPC-b-PDMA macro-CTAs. MPC (5.537 g, 18.75 mmol), CADB (210.0 g, 0.75 mmol), V-501 (42.0 mg, 0.15 mmol), 5.0 w/w % aqueous NaHCO3 solution (5.040 g), and deionized water (26.081 g) were added to a Schlenk flask with a magnetic stir bar. This reaction mixture was then stirred until all the CADB had dissolved in an icebath. The solution was purged using nitrogen gas for approximately 30 min and then placed in a pre-heated oil bath at 70 °C. The polymerization was terminated after 120 min via rapid cooling in an ice bath and exposure to air. The crude PMPC macro-CTA was then purified by dialysis against deionized water using semi-permeable cellulose tubing (SPECTRA/POR, molecular weight cut-off of 1000Da) with at least six changes of deionized water for 24 h, followed by freeze-drying. The resulting PMPC25 macro-CTA (3.661 g, 0.496 mmol), V-501 (28.0 mg, 0.10 mmol), DMA (2.339 g, 14.88 mmol), and dehydrated methanol (14g, Wako) were added to a Schlenk flask with a magnetic stir bar. The solution was purged using nitrogen gas for approximately 30 min and then placed in a pre-heated oil bath at 60 °C. The second polymerization was terminated after 60 min via rapid cooling in an ice bath and exposure to air. The crude PMPC25-b-PDMA macro-CTA was then purified by dialysis and freeze-drying in the same way as PMPC macro-CTA. Each actual degree of polymerization (DP) of PMPC and PMPC-b-PDMA macro-CTA was 25 and 4, respectively, as judged by 1H NMR spectroscopy, which indicates that the CTA efficiency is close to 100 %. Synthesis of PMPC-b-PDMA-b-PHPMA via RAFT Aqueous Dispersion Polymerization The resulting PMPC25-b-PDMA4 macro-CTA (36.6−61.0 mg, 4.57×10-3−7.61×10-3 mmol, V-501 (0.51−0.85 mg, 1.82×10-3−3.03×10-3 mmol) and varying amounts of HPMA (263.4−439.0 g, 1.83−3.05 mmol; target DP 100−400), and deionized water (1.7−1.5 g) for 15-25 w/w% solids concentration in a Schlenk flask with a magnetic stir bar. These solutions were stirred in an ice-bath until all reagents had dissolved. After purging using nitrogen gas for approximately 30 min, each polymerization was carried out at 70 °C and then quenched after 24 h via rapid cooling in an ice bath and exposure to air. 1

H NMR Spectroscopy. The 1H NMR spectra for detailed structures were recorded on JEOL

JNM-ECX500 (500 MHz) spectrometers. In the case of block copolymer solution, the resulting


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assemblies in water after polymerization was directly diluted by either D2O or d4-methanol to ca. 1 w/w% before measurement. Gel permeation chromatography (GPC). Molecular weight distributions (MWDs) were obtained by GPC using a Tosoh CCPM-II pump and two Polymer Laboratories PL Gel 5 µm Mixed-C (7.5 × 300 mm) columns in series with a PL Gel guard column at 40 °C connected to a Tosoh RI-8012 refractive detector. The eluent was a 3:1 v/v % chloroform/methanol mixture containing 2 mM LiBr at a flow rate of 1.0 mL min-1. The number-average molecular weight (Mn) and polydispersity index (Mw/Mn) were calculated from GPC curves using poly(methyl methacrylate) (PMMA) calibration standards. Dynamic Light Scattering (DLS). DLS studies of 0.5 w/w% assemblies were performed using a Zetasizer Nano-ZSP instrument (Malvern Instruments) at 25 ºC at the backscatter angle of 173º. The mean particle diameter (Dh) and polydispersity index (PDI) of the assembles were calculated by cumulants analysis of the experimental correlation function using Zeta Software version 7.04. The results were averaged over nine consecutive runs. Dynamic Mode Atomic Force Microscopy (DM-AFM). AFM images for block copolymer assemblies prepared were recorded under ambient temperature using the dynamic mode with an SPM-9700 (Shimadzu) scanning probe microscope. A silicon AFM probe tip (Olympus, OMCLAC240TS) was used, which was with a radius of 7 nm, a resonance frequency of 70 kHz, and a spring constant of 2 N/m. The samples for AFM imaging were prepared by placing a 20 µL drop of the resulting assemblies in water (ca. 0.05 wt%) on freshly cleaved muscovite mica (ca. 1 cm × 1 cm, V-4 grade, Alliance Biosystems) and dried in air for a day. Transmission Electron Microscopy (TEM). TEM studies were conducted using a JEOL JEM2100 instrument operating at 200 kV with a Gatan ORIUS SC200D CCD camera. To create a hydrophilic surface, copper grids with carbon-coated support film (ELS-C10, Okenshoji) were glow discharged for 15 s. Ca. 5 µL of block copolymer assemblies in water (0.5 wt%) was put on the grid for 30 s and then blotted with filter paper. The grid was immersed for 60 s in EM stainer (Nisshin EM) for negative staining. The stained grid was then blotted with filter paper and dried in air. Ag-NPs loaded assemblies in water was directly put on the grid for 30 s and then blotted with filter paper without staining. Synthesis of Ag-NPs Loaded PMPC25-b-PDMA4-b-PHPMA400 Assemblies in Water and Their Catalytic Performance. The resulting PMPC25-b-PDMA4-b-PHPMA400 assemblies in water (241.6 mg) was diluted with 574.8 mg of water and then added in to a flask with a magnetic stir bar. 1.0 % AgNO3 (Wako) aqueous solution (50.0 mg) was added to the flask with stirring for 30 min. The mixture was then reduced with 0.1% NaBH4 aqueous solution (133.6 mg). And then, the resulting Ag-NPs loaded PMPC25-b-PDMA4-b-PHPMA400 assemblies in water were further diluted 100 times with water: [AgNO3]/[DMA unit]/[NaBH4] = 1:1:1.5 molar ratio. Another NaBH4


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aqueous solution (1 mL, 100mM) and p-NP (1.5 mL, 0.1mM) were directly mixed into a quartz cell. The 0.05 mL of the as-prepared Ag-NPs loaded PMPC25-b-PDMA4-b-PHPMA400 assemblies in water was introduced into the quartz cell at 25 °C. The catalytic performance was monitored by a JASCO V-500 UV/vis spectrometer with a Peltier-type thermostatic cell holder ETC-505.

Results and Discussion Synthesis of PMPC and PMPC-b-DMA Macro-CTAs. The synthetic route used in this work to obtain PMPC-b-PDMA-b-PHPMA is outlined in Figure 1. This is based on the previous report of dispersion polymerization for PMPC25-b-PHPMAm diblock copolymer self-assemblies.32 In the report, PMPC macro-CTA was deployed as a reactive steric stabilizer for diblock copolymer nanoparticles via RAFT dispersion polymerization. The PMPC macro-CTA was chain-extended using a water-soluble monomer of HPMA at 70 ºC. Since the polymer, PHPMA, was water-insoluble, the growing PHPMA block led to PISA to form spheres, worms, vesicles, and other mixed phases. Furthermore, the morphology could be targeted by simply changing the total solids concentration. In this work, PMPC and PMPC-b-PDMA macro-CTA were first synthesized by RAFT polymerization (solution polymerization) to impart functionality to PMPC25-b-PHPMAm. According to the literature,32 near-monodisperse PMPC macro-CTA was synthesized using V-501 initiator and CADB as a RAFT agent in water in the presence of NaHCO3 (5 w/w% aqueous NaHCO3 solution): [MPC]0 = 15 w/w%, [MPC]0:[CADB]0:[V-501]0 = 25:1:0.2 molar ratio. The polymerization proceeded to full conversion in 2 h, which was determined by 1H NMR analysis of the diluted reaction mixture using D2O. As-synthesized PMPC was purified by dialysis against deionized water, followed by freeze-drying. The detailed structure and DP of the final PMPC macro-CTA product were determined by 1H NMR spectroscopy (Figure 2A). The Mn and Mw/Mn of the PMPC macro-CTA were 7600 and 1.08 calculated from GPC curves on the basis of a PMMA calibration curve (Figure 3B, the top MWD). The actual DP of 25 was estimated by comparing the integrations of the aromatic RAFT end group (a) and the three quaternary nitrogen methyl groups (b) or methylene protons (c) in the PMPC chains, which was in good agreement with the targeted DP. Next, the resulting PMPC chain was extended using DMA with V-501 in methanol at 70 ºC ([DMA]0:[PMPC25]0:[V-501]0 = 30:1:0.2 molar ratio, solids concentration = 30.0 w/w%). Here, methanol was used as the solvent instead of water for the synthesis of PDMA because PDMA exhibits LCST-type thermoresponsive behavior in water around 50 ºC.55 The DMA polymerization proceeded to 13.6% conversion in 1 h, determined by 1

H NMR analysis of the diluted reaction mixture in d4-methanol. The resulting copolymer was


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easily soluble in water and was purified by dialysis against deionized water at room temperature to remove DMA residues, followed by freeze-drying. The obtained copolymer MWD was shifted to higher molecular weights relative to the PMPC25 macro-CTA while keeping the Mw/Mn value (Figure 3B, the second MWD). The Mn and Mw/Mn of the PMPC macro-CTA were 8400 and 1.08, respectively. This result indicated adequately high blocking efficiency. Furthermore, the aromatic RAFT end group remained in the polymer without decomposition (h), and the actual DP of PMPC and PDMA were determined to be 25 and 4, respectively, on the basis of the integrations of the respective methyl groups of PMPC (g) and PDMA (i) via 1H NMR analysis in Figure 2B.

Figure 2. 1H NMR spectra of (A) PMPC25 and (B) PMPC25-b-PDMA4 macro-CTAs in D2O at 20 ºC. The insets are the chemical structures with full peak assignments.


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Figure 3. (A) Variation of Mn and Mw/Mn with targeted DP of HPMA for the resulting PMPC25-b-PDMA4-b-PHPMAz (z = 100-400) triblock copolymer assemblies (entries 1-8) and (B) MWDs for aqueous dispersion polymerization of HPMA at 70 ºC (entries 1-8) and for the related PMPC25 and PMPC25-b-PDMA4 macro-CTAs: [PMPC25-b-PDMA4]0/[V-501]0/[HPMA]0 = 1/0.4/100-400 molar ratio, total solids concentration = 25.0 w/w% (= [PMPC25-b-PDMA4 (g) +HPMA(g)]/[all reaction mixtures (g)]×100).

Synthesis of PMPC25-b-DMA4-b-PHPMAz Assemblies via RAFT Aqueous Dispersion Polymerization at 25 w/w% Solids Concentration. The PMPC25-b-PDMA4 macro-CTA was further chain-extended with HPMA via RAFT aqueous dispersion polymerization. Since dispersion polymerization of HPMA reached full conversion in a day against all conditions, the polymerizations were carried out for 24 h by changing the feed ratio of HPMA and the solids concentration. The solids concentration is defined as 100 × [PMPC25-b-PDMA4 (g) + HPMA (g)]/[all reaction mixture (g)]. The HPMA is expected to be the core monomer for PMPC25-b-PDMA4-stabilized self-assembly in the case of the RAFT aqueous dispersion polymerization of HPMA. As the HPMA chain grows from the water-miscible PMPC25-b-PDMA4 macro-CTA, it reaches a critical DP at a certain point and becomes sufficiently hydrophobic such that micellar nucleation is induced. Here, the targeted DP of HPMA, i.e., [HPMA]0/[PMPC25-b-PDMA4]0, was set from 100 to 400. During the course of the dispersion polymerization, the polymerization mixture changed from transparent to opaque. This indicates that both the progress of the polymerization and in situ self-assembly were occurred by micellar nucleation of HPMA. Finally, the polymerization


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reached full monomer conversion of HPMA and took on the final morphology. The polymerization-induced self-assembled morphologies were confirmed by means of DM-AFM. Table 1 summarized the representative polymerization results including morphologies. In entries 1-8, a series of PMPC25-b-PDMA4-b-PHPMAz triblock copolymer nanoparticles with varying target DPs (= z) of PHPMA were synthesized at 25 w/w% solids concentration in water. All the polymerizations for various target DPs of PHPMA had good control over the polymer chain growth. GPC analysis indicated near-monodispersity and high blocking efficiency, since there was minimal contamination of the triblock copolymer chains with unreacted PMPC25-b-PDMA4 macro-CTA (Figure 3B). Thus, this aqueous dispersion polymerization system seems to be a well-controlled polymerization system. Hence, the resulting Mn is directly proportional to the targeted DP as seen in Figure 3A. In particular, the resulting Mw/Mn values remain below 1.15 even when the targeted DP of HPMA is higher, such as in PMPC25-b-PDMA4-b-PHPMA400. Given that the HPMA monomer sometimes contains dimethacrylate impurities due to transesterification,56 this indicates that little cross-linking occurred thanks to the Lewis basic PDMA block.

Table 1. Representative Molecular Characterization Data of PMPC25-b-PDMA4-b-PHPMAz Block Copolymers Prepared by RAFT Aqueous Dispersion Polymerization Using PMPC25-b-PDMA4 Macro-CTA at 70 °C a targeted DP of

solids

Dh

Mn

Polymer Structureb

entry

HPMAc

(w/w %)d

(kg/mol)e

Mw/Mne

PDIf

Morphologyg

(nm)f

1

PMPC25-b-PDMA4-b-PHPMA100

100

25

2.07

1.08

25

0.06

spheres

2


150

25

3.10

1.08

33

0.13

spheres

3


200

25

3.97

1.09

57

0.28

spheres

4


250

25

4.59

1.09

309

0.28

worms

5


300

25

5.27

1.12

589

0.30

worms

6


325

25

5.50

1.15

753

0.29

jellyfish

7


350

25

5.85

1.15

359

0.20

worms/vesicles

8


400

25

6.08

1.15

117

0.08

vesicles

9


400

20

6.25

1.17

268

0.26

worms


400

15

6.00

1.20

51

0.08

spheres

10 a

[PMPC25-b-PDMA4]0/[V-501]0/[HPMA]0 = 1/0.4/100-400 molar ratio, total solids concentration = 25.0 w/w%. The segment DP in the formula was determined by 1H NMR spectroscopy on the basis of PMPC25-b-PDMA4 macro-CTA. c [HPMA]0/[PMPC25-b-PDMA4]0 d 100 × [PMPC25-b-PDMA4 (g) + HPMA (g)]/[all reaction mixtures (g)]. e Determined by GPC [poly(methyl methacrylate) (PMMA) standards, 3:1 CHCl3/methanol eluent with 2 mM LiBr]. f Determined by DLS measurement at 25 ºC. g By DM-AFM. b


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To confirm the in situ formation of PHPMA-core aggregates in water, typical block polymers obtained in the RAFT aqueous dispersion polymerization were directly diluted in either a selective solvent of D2O or a good solvent of d4-methanol, and 1H NMR spectra were measured as shown in Figure 4A and B, respectively. Compared to the signals of PMPC25-b-PDMA4 macro-CTA in Figure 2B, only signals from the PMPC25-b-PDMA4 moiety were observed in the spectrum recorded in D2O at 20 ºC (Figure 4A). None or broader PHPMA signals were visible in D2O. In contrast, all the proton signals expected for the PMPC, PDMA, and PHPMA blocks were visible in the 1H NMR spectrum recorded in d4-methanol in Figure 4B, since this is a good solvent for both blocks. These 1H NMR observations suggest that the PMPC25-b-PDMA4 chains act as the reactive and solvated steric stabilizer (shell), while the PHPMA chains form the non-solvated micelle core, i.e., three-layer onion micelles could be obtained with the PMPC chains in the outermost layer. In addition, the resulting DPs of PHPMA were determined in d4-methanol and are also summarized in Table 1.

Figure 4. Typical 1H NMR spectra at 20 ºC of PMPC25-b-PDMA4-b-PHPMA300 (entry 5) prepared via RAFT aqueous dispersion polymerization, diluted in (A) D2O or diluted in (B) d4-methanol.


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DLS studies were conducted to check the mean particle diameter and the polydispersity index (PDI)

for

entries

1-8.

The

representative

DLS

size

distributions

for

various

PMPC25-b-PDMA4-b-PHPMAz (z = 100-400) diblock copolymer assemblies are shown in Figure 5. As the DP of PHPMA increased from 100 to 325, the DLS size distribution shifted toward the larger diameter region. The mean particle diameter (hydrodynamic diameter, Dh) increased in a nearly linear fashion with the increasing targeted DP of HPMA up to 200. The calculated values of PDI were relatively narrow (PDI ≤ 0.28). When the chain length of the core-forming block increased, larger micelles were always obtained. This is essentially the same aggregation behavior as that in the post-aggregation using the block copolymer in a selective solvent.39,40,42 However, at targeted DP ≥ 250, especially DP = 250-350 (entries 4-7), multimodal distributions were obtained. At DP = 400, assemblies with relatively larger size than those at DP = 100-200 but smaller than those at DP 250-350 were obtained. Thus, we predicted that these assemblies (DP ≥ 250) were not systematically spherical micelles via DLS analysis.

Figure 5. DLS particle size distributions (volume vs. mean particle diameter) obtained for the same series of triblock copolymer assemblies of PMPC25-b-PDMA4-b-PHPMA100-400 prepared at 25 w/w% solids concentration via RAFT aqueous dispersion polymerization of HPMA using PMPC25-b-PDMA4 macro-CTA. The inset numbers 1-8 indicate entries in Table 1.

For the assemblies of entries 1-8 excluding entry 6, the AFM images are shown in Figure 6.


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From DM-AFM studies on freshly cleaved muscovite mica under the dry condition, the resulting self-assemblies showed spheres (entries 1-3), worms (entries 4 and 5), worms including vesicles (entry 7), and vesicles (entry 8) according to the targeted DP of HPMA. For all the samples, the AFM sizes were smaller than the DLS sizes, regardless of their morphologies. For example, for the spherical micelle of entry 3, the mean particle diameter is 33 nm from the AFM image in Figure 6C, but 57 nm from DLS analysis. This indicates that PMPC25-b-PDMA4 shells are sufficiently spreading or swelling in water. Comparing entries 4 and 5 in Figure 6D and E, respectively, the mean particle diameter for entry 5 was larger than that of entry 4, increasing from 44 nm to 50 nm, and the length of the worm for entry 5 (ca. > 5 µm) also appeared to be much longer than that of entry 4, judging from AFM analysis. Since the core PHPMA is known to be fully-stretched in the case of PMPC-b-PHPMA worms in water, the mean diameter of the worms of PMPC-b-PDMA-b-PHPMA is likely to depend on the DP of PHPMA. Furthermore, for entry 7 in Figure 6F, it is observed that both vesicles and worms were formed, and the worms were partly swirled and in a state similar to a vesicle. In the case of vesicles, the AFM topography (height) measurements also indicated flattening on mica (33 nm height in Figure 8G) and dimpling in the middle due to the AFM observation under dry condition. The dimple in the vesicles can be observed by cross-sectional profile of the resulting AFM image in Figure 6G (right).

Figure 6. Representative DM-AFM (height) images of PMPC25-b-PDMA4-b-PHPMAz


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assemblies for entries 1-5, 7, and entry 8 with cross-sectional profiles.

Interestingly, anisotropic jellyfishes were observed in the AFM image for entry 6 as an assembly between vesicles and worms (Figure 7A). This is the predicted morphology for the transition to vesicle formation, which supports the assembling behavior via RAFT dispersion polymerization proposed by Armes et al.57,58 In view of the packing parameter P, the jellyfish morphology is clearly unstable. Thus, this observation suggests that the triblock PMPC-b-PDMA-b-PHPMA copolymer assemblies obtained via this RAFT aqueous dispersion polymerization system are kinetically frozen in the used solvent, i.e., water. Therefore, similar to other assemblies for entries 1-5 and 7-8, there would be no change in morphology in the same solvent, i.e., water, even if the concentration changed. TEM measurements by negative staining confirmed that there were many jellyfish formations in which the worm chains grew and contracted (Figure 7B). In order to maintain such a mysterious nano-organization in good solvent, we attempted quaternization between DMA units by adding 1,2-bis(2-iodoethoxy)ethane (BIEE) and cross-linking of the inner shell. The quaternization proceeded smoothly without changing the jellyfish morphology in the same solvent of water. However, it was difficult to maintain the same morphology in 1:1 (w/w) water/methanol mixture including good solvent of methanol because partially-collapsed short worms were formed (the results are shown in Figures S1 and S2, Supporting Information). This suggested that the triblock copolymer assembly was present in a quite narrow packing parameter P region.

Figure 7. (A) DM-AFM (height) and (B) TEM images of jellyfish morphology for entry 6.

PMPC25-b-DMA4-b-PHPMA400 Assemblies Synthesized under Various Solids Concentrations and their Templates for Ag-NPs. In RAFT aqueous dispersion polymerization, various


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morphologies can be formed depending on the polymerization parameters as well as the chain length, and it has been shown that the final copolymer concentration can also profoundly affect the assembly morphology. Hence, different morphologies using identical and kinetically-frozen block copolymers can be obtained in water in this ABC triblock copolymer system. As expected, when the target DP of PHPMA is set at 400 for RAFT aqueous dispersion polymerization with the various solids concentrations, the resulting Mn and Mw/Mn for the final triblock copolymer products were almost the same, regardless of the different solids concentration (Figure 8A). Significantly, the resulting morphologies were clearly different despite the same PMPC25-b-PDMA4-b-PHPMA400 assemblies. Dilution with water or concentration by centrifugation did not lead to any change in the block copolymer morphology, as confirmed by DM-AFM and DLS measurements. This indicates that the resulting assemblies in water are kinetically frozen. The representative DLS size distributions of diluted 0.5 w/w% assemblies in water prepared at 15, 20, or 25 w/w% solids concentration are shown in Figure 8B. As the solids concentration decreased in the polymerization, DLS size distributions changed from monomodal (entry 8, Dh = 117 nm, PDI = 0.08) to monomodal but smaller (entry 10, Dh = 51 nm, PDI = 0.08) through multimodal and larger (entry 9, Dh = 268 nm, PDI = 0.26). These results are also summarized in Table 1. In practice, vesicles, worms, or spheres were observed for entries 8, 9 and 10, respectively, via AFM images in Figure 6G (entry 8) and Figure 9A (entry 9) and 9B (entry 10). Using the resulting Dh values and AFM images, the surface area ratio per particle can be simply calculated as follows: vesicle: worm: sphere = 5.3:20.1:1, where the vesicle was treated as a sphere and the worm was calculated as a cylinder with a diameter of 51 nm and a length of 1 µm. Thus, the production of vesicles, worms, and spheres can be tuned and enables the facile preparation of various self-assemblies by directly changing the solids concentration in polymerization.


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Figure 8. (A) GPC curves for targeted PMPC25-b-PDMA4-b-PHPMA400 prepared via RAFT aqueous dispersion polymerization of HPMA using PMPC25-b-PDMA4 macro-CTA at 70 ºC at 25, 20, and 15 w/w% solids concentration for entries 8, 9, and 10, respectively: [PMPC25-b-PDMA4]0/[V-501]0/[HPMA]0 = 1/0.4/400 molar ratio. (B) DLS particle size distributions (volume vs. mean particle diameter) of PMPC25-b-PDMA4-b-PHPMA400 assemblies in water.


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Figure 9. DM-AFM (height) images of targeted PMPC25-b-PDMA4-b-PHPMA400 assemblies: (A) worms of entry 9 and (B) spheres of entry 10. For vesicles (entry 8), see Figure 6G. TEM images of all the targeted PMPC25-b-PDMA4-b-PHPMA400 assemblies for entries 8-10 loaded with Ag-NPs (C-E) and the corresponding particle size distributions of Ag-NPs (C’-E’). These Ag-NPs loaded assemblies (C-E) were prepared with [AgNO3]/[DMA unit]/[NaBH4] ratios of 1:1:1.5 at 25 ºC.

Using the obtained three PMPC25-b-PDMA4-b-PHPMA400 assemblies as templates, Ag-NPs were obtained through in situ reduction of AgNO3 via the electrostatic interactions between Ag+ ions and amine groups of PDMA. The in situ chemical reduction of Ag+ ions with NaBH4 (eq. 2) led to elemental metallic nanoparticles confined within the PMPC25-b-PDMA4-b-PHPMA400 scaffolds: 2AgNO3 + 2NaBH4 + 6H2O → 2Ag + 2NaNO3 + 2B(OH)3 + 7H2


(2)

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The reduction from Ag(I) to Ag(0) can be followed by UV/vis absorption spectroscopy (Figure 10). The diluted PMPC25-b-PDMA4-b-PHPMA400 assemblies in water (0.1 w/w%; aqueous 1.52 mM polymer solution) were transparent. Within a minute after the addition of 0.1 w/w% NaBH4 aqueous solution, the solution became pale brown and an absorption band (λmax) was observed at 407 nm, attributed to the surface plasmon resonance characteristic of the metallic Ag-NPs. The resulting solution maintained excellent colloidal stability over one month without any precipitation, regardless

of

assembly

morphology

(entries

8-10).

This

result

indicates

the

PMPC25-b-PDMA4-b-PHPMA400 assemblies act as stabilizers for Ag-NPs. TEM images of the resulting Ag-NPs loaded PMPC25-b-PDMA4-b-PHPMA400 prepared with [AgNO3]/[DMA unit]/[NaBH4] ratios of 1:1:1.5 are shown in Figure 9C-E. These TEM images could be easily obtained without staining because there were many Ag-NPs inside the respective assemblies, regardless of assembly morphology. Since all the assembly scaffolds used here were exactly the same triblock copolymer and the same amount of AgNO3 was loaded on them, it is possible to consider the relationship between the surface area and almost the same-sized Ag-NPs (~3 nm) present (Figure 9C’-E’). From the TEM results and original AFM images, the worms obviously have the largest surface area, followed by vesicles and spheres as mentioned above. We next examined the catalytic activity using these Ag-NPs loaded PMPC25-b-PDMA4-b-PHPMA400 assemblies.

Figure 10. UV/vis spectra at ambient temperature of (A) as-synthesized diluted PMPC25-b-PDMA4-b-PHPMA400 assemblies in water (entry 10), (B) the assemblies in water after

loading

AgNO3,

and

(C)

followed

by

reduction

with

NaBH4:

[PMPC25-b-PDMA4-b-PHPMA400] = 1.52 mM (0.1 w/w%), [AgNO3]/[DMA unit]/[NaBH4] ratios


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of 1:1:1.5 at 25 ºC.

In general, p-NP cannot be reduced to p-aminophenol (p-AP) with NaBH4 without a silver catalyst, and the applicability of Ag-NPs as catalysts depends on their size, surface area, and stability.59-63 Thus, the reduction of p-NP to p-AP was conducted in a quartz cell in the presence of as-prepared Ag-NPs loaded PMPC25-b-PDMA4-b-PHPMA400 assemblies in water (samples of Figures 9C-E), aqueous p-NP solution, and aqueous NaBH4 solution with stirring of 100 rpm. The reduction reaction was conducted at [p-NP]0:[Ag-NPs] = 1:1 molar ratio in the presence of NaBH4, and the final concentrations of p-NP and NaBH4 were set at 0.15 µM and 0.1 mM, respectively. As shown in Figure 11A, the absorption peak at 400 nm decreased continuously and a new absorption peak around 300 nm increased gradually. This indicates the successful reduction of p-NP to p-AP (eq. 3). The reaction was complete in 35-80 min, depending on the PMPC25-b-PDMA4-b-PHPMA400 assembly morphology.

(3)

To compare the catalytic activity of Ag-NPs in each PMPC25-b-PDMA4-b-PHPMA400 assembly, the conversion of p-NP to p-AP was determined by the plot of ln(A0/At) vs. time (Figure 11B), where At is the UV absorbance at 400 nm and A0 is the initial UV absorbance at 400 nm after addition of NaBH4. The progress of reaction can be read directly from the UV absorbance curves in Figure 11A for worms and Figure S3 for vesicles and spheres. Since ln(A0/At) is proportional to time, regardless of PMPC25-b-PDMA4-b-PHPMA400 assembly morphologies, the slope of the curve represents the apparent first-order rate constant (kapp). The kapps estimated from the plots are 2.07 × 10-3 s-1, 2.62 × 10-3 s-1, and 0.95 × 10-3 s-1 for vesicles (entry 8), worms (entry 9), and spheres (entry10), respectively. Thus, the order of rate constants for reduction was worms > vesicles > spheres, which is in good agreement with the order of the predicted surface areas of the assemblies of PMPC25-b-PDMA4-b-PHPMA400 in Figure 9C-E.


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Figure 11. (A) Typical UV/vis spectra of p-NP reduced with NaBH4 catalyzed with Ag-NPs loaded PMPC25-b-PDMA4-b-PHPMA400 worms (entry 9) in water [see Figure S3 and S4 for PMPC25-b-PDMA4-b-PHPMA400 vesicles (entry 8) and spheres (entry 10), respectively], and (B) the relationships between ln(A0/At) and time for Ag-NPs loaded PMPC25-b-PDMA4-b-PHPMA400 vesicles (blue), worms (red), or spheres (black). The slope corresponds to the apparent first-order rate constant (s-1).

In conclusion, biomimetic ABC triblock copolymers of PMPC-b-PDMA-b-PHPMA were synthesized by RAFT aqueous dispersion polymerization of HPMA using a PMPC-b-PDMA macromolecular chain transfer agent (macro-CTA). This formulation enabled the in situ synthesis of three-layer onion micelles with an outer shell of PMPC, an inner shell of PDMA, and a PHPMA core. In the synthesis of PMPC25-b-PDMA4-b-PHPMAz at a constant 25 w/w% solids concentration, the resulting assemblies changed from spheres to worms to jellyfishes to vesicles when the targeted PHPMA chain length increased from 100 to 400mer at full monomer


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conversion. In the synthesis of identical PMPC25-b-PDMA4-b-PHPMA400 copolymers, the assembly morphology could be controlled from vesicles to spheres through worms by varying the solids concentration in the polymerization from 25 to 15 w/w% at full monomer conversion. Using the resulting three PMPC25-b-PDMA4-b-PHPMA400 assemblies as a scaffold, Ag-NPs were obtained. The resulting Ag-NPs loaded in various assemblies exhibited excellent stability, dispersibility and catalytic activity for the reduction of p-NP. The order of rate constant for reduction using the Ag-NPs loaded in the assemblies was worms > vesicles > spheres, which corresponds to the order of surface areas of the assemblies of PMPC25-b-PDMA4-b-PHPMA400. Hence, worms were confirmed to be the best Ag-NPs scaffold. These results can be achieved thanks to the kinetically-frozen PMPC25-b-PDMA4-b-PHPMA400 assemblies with identical compositions.

Acknowledgment We express our gratitude to Prof. Satoshi Irie for TEM measurement.

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Table of Contents Graphic


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Synthesis and Nano-object Assembly of Biomimetic Block Copolymers

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