RAFT Aqueous Dispersion Polymerization Yields Poly(ethylene glycol)-Based Diblock Copolymer Nano-Objects with Predictable Single Phase Morphologies

A poly(ethylene glycol) (PEG) macromolecular chain transfer agent (macro-CTA) is prepared in high yield (>95%) with 97% dithiobenzoate chain-end functionality in a three-step synthesis starting from a monohydroxy PEG113 precursor. This PEG113-dithiobenzoate is then used for the reversible addition–fragmentation chain transfer (RAFT) aqueous dispersion polymerization of 2-hydroxypropyl methacrylate (HPMA). Polymerizations conducted under optimized conditions at 50 °C led to high conversions as judged by 1H NMR spectroscopy and relatively low diblock copolymer polydispersities (Mw/Mn < 1.25) as judged by GPC. The latter technique also indicated good blocking efficiencies, since there was minimal PEG113 macro-CTA contamination. Systematic variation of the mean degree of polymerization of the core-forming PHPMA block allowed PEG113-PHPMAx diblock copolymer spheres, worms, or vesicles to be prepared at up to 17.5% w/w solids, as judged by dynamic light scattering and transmission electron microscopy studies. Small-angle X-ray scattering (SAXS) analysis revealed that more exotic oligolamellar vesicles were observed at 20% w/w solids when targeting highly asymmetric diblock compositions. Detailed analysis of SAXS curves indicated that the mean number of membranes per oligolamellar vesicle is approximately three. A PEG113-PHPMAx phase diagram was constructed to enable the reproducible targeting of pure phases, as opposed to mixed morphologies (e.g., spheres plus worms or worms plus vesicles). This new RAFT PISA formulation is expected to be important for the rational and efficient synthesis of a wide range of biocompatible, thermo-responsive PEGylated diblock copolymer nano-objects for various biomedical applications.


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dichloromethane (approximately 200 mL) was gradually added until the solution became clear. Triethylamine (2.13 mL, 1.52 g 15.0 mmol) was added dropwise with stirring, followed by the dropwise addition of mesyl chloride (1.71 g, 1.18 mL, 15.0 mmol). After 18 h, the reaction solution was filtered to remove the insoluble white triethylamine hydrochloride salt, followed by precipitation into excess diethyl ether.
The off-white PEG 113 -OMs product (47 g) was isolated by filtration and dried under vacuum.
The PEG 113 -Ms intermediate (45.0 g) was added to a 25% aqueous ammonia solution (300 mL) in a 1 L duran bottle. The lid was tightly sealed with the aid of Parafilm R and the reaction solution was stirred for four days at 20 o C. The lid was subsequently removed and the ammonia was allowed to evaporate slowly over three days at the back of a fumehood. NaOH (5 M) was added dropwise to the solution until the pH reached 13 and the polymer was extracted into dichloromethane (3 x 100 mL). The combined organics were washed with brine and subsequently dried over anhydrous magnesium sulfate. After concentrating under vacuum, the crude PEG 113 -NH 2 product was precipitated into excess diethyl ether and dried under vacuum to produce PEG 113 -NH 2 (43.0 g). The presence of the primary amine end-group was indicated by a triplet at δ 2.9 ppm by 1 H NMR spectroscopy, which corresponds to the two methylene protons adjacent to the amine. Comparison of this integrated triplet with that of the peaks corresponding to the protons on the PEG backbone indicated more than 98% amine end-group functionality.

Synthesis of poly(ethylene glycol) dithiobenzoate (PEG 113 -DB) macro-CTA
Before carrying out the reaction, all glassware was rigorously dried at 120°C overnight to remove all traces of water. SCPDB (3.50 g, 9.3 mmol) was dissolved in anhydrous dichloromethane (20 mL) in a 100 mL two-neck round-bottomed flask fitted with a dropping funnel. A solution of dried PEG 113 -NH 2 (39.5 g, 7.9 mmol) in dichloromethane (100 mL) was added dropwise to the SCPDB solution over a period of approximately 1 h. This reaction solution was stirred overnight, precipitated into excess diethyl ether and the resulting pink polymer (PEG 113 -DB) was analyzed by 1 H NMR spectroscopy. The

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presence of aromatic signals at δ 7.4 -7.9 confirmed the presence of the desired terminal dithioester group and comparison of these integrated signals with that at δ 3.0 − 4.0 due to the ethylene glycol protons of the PEG chain suggested approximately 93 % functionalization. Visible spectroscopy was also used to determine the dithiobenzoate content of the PEG chains, using a calibration curve constructed from a series of methanolic solutions containing varying concentrations of CPADB. From this plot, a molar extinction coefficient of 104 800 ± 640 L mol -1 cm -1 was calculated for the absorption maximum observed at 516 nm in methanolic solution (see Figure S3). Using this information, a mean degree of dithiobenzoate functionalization of 97 ± 2 % was calculated for three separate PEG 113 -DB batches.

RAFT dispersion polymerization of HPMA using the PEG 113 -DB macro-CTA
The following representative protocol was used for the synthesis of PEG 113 -PHPMA 300 at 10 % w/w solids using AIPD initiator at 50°C. AIPD initiator (0.0025 g, 0.0077 mmol), PEG 113 -DB macro-CTA (0.1225 g, 0.0231 mmol; [CTA]/[AIPD] molar ratio = 3.0) and HPMA monomer (1.00 g, 6.94 mmol, target DP = 300) were weighed into a roundbottomed flask containing a magnetic stir bar. These reagents were dissolved in previously deoxygenated water (10.13 mL, 10% w/w) and purged with nitrogen for 30 minutes. The flask was sealed using a rubber septum under a positive nitrogen flow and immersed in an oil bath at 50°C. For the kinetic studies, aliquots were periodically removed for analysis by 1 H NMR spectroscopy and GPC. When the polymerization had proceeded for 2.5-3 h, the reaction was quenched by exposure to air and cooling to 20 o C.

Dynamic light scattering (DLS)
DLS measurements were conducted at 25 o C using a Malvern Instruments Zetasizer Nano series instrument equipped with a 4 mW He-Ne laser operating at 633 nm, an avalanche photodiode detector with high quantum efficiency, and an ALV/LSE-5003 multiple tau digital correlator electronics system. Light scattering was detected at 173 o S5 and hydrodynamic diameters were determined using the Stokes-Einstein equation, which assumes spherical, non-interacting, perfectly monodisperse particles.
Nuclear magnetic resonance spectroscopy 1 H NMR spectra were acquired using either a Bruker 250 MHz or a 400 MHz spectrometer.
Samples were dissolved in CDCl 3 , CD 2 Cl 2 or CD 3 OD at 10-20 mg mL -1 . All chemical shifts are reported in ppm (δ). The average number of scans accumulated per spectrum was typically 64.

Visible absorption spectroscopy
A PC-controlled Perkin-Elmer Lambda 25 spectrophotometer was used for recording spectra at 20 o C from 400 nm to 700 nm at a scan rate of 240 nm min -1 and a slit width of 1 nm.

Gel permeation chromatography (GPC)
The GPC set-up comprised two 5 µm Mixed-C columns; a WellChrom K-2301 refractive index detector operating at 950 ± 30 nm and a Precision detector PD 2020 light scattering detector (with scattering angles of 90º and 15º). THF eluent contained 2.0% v/v triethylamine and 0.05% w/v butylhydroxytoluene (BHT) and a flow rate of 1.0 mL min -1 was employed. A series of ten near-monodisperse poly(methyl methacrylate) standards (M p ranging from 1,280 to 330,000 g mol -1 ) were employed as calibration standards in conjunction with the above refractive index detector.

Transmission electron microscopy (TEM)
Copper/palladium TEM grids (Agar Scientific) were surface-coated in-house to produce a thin film of amorphous carbon. The grids were then plasma glow-discharged for 30 seconds to create a hydrophilic surface. After glow discharge, the grids were immersed in 0.10% w/v aqueous dispersions containing the diblock copolymer nano-objects for 40 S6 seconds. After blotting to remove excess sample dispersion, the grids were negatively stained by immersion in uranyl formate solution (0.75% w/v) for 30 seconds. The grid was blotted again to remove excess stain and dried using a vacuum hose. This heavy metal salt acts as a negative stain to improve electron contrast. Imaging was performed using a FEI Tecnai Spirit TEM instrument equipped with a Gatan 1kMS600CW CCD camera operating at 120 kV. is the length of the scattering vector and θ is a half of the scattering angle) or one camera length setup (3 m, q range 0.03 -0.2 nm -1 ) was used for the data collection. A solution cell with S7 removable mica windows was used as a sample holder. The sample was poured into the solution cell and a mica window was placed on top. This loading protocol avoided shear effects, which could otherwise occur if the sample was injected into the cell. A multiframe approach with a frame rate of 10 Hz was used for SAXS data collection. This acquisition method enables stability of samples exposed to X-ray beam to be monitored. It has been noticed that in some cases a long exposure time (for more than 100 ms) to the X-ray beam, heating the sample locally, couses changes in the SAXS pattern. Scattering data reduced by SAXS utilities software package (integration, normalization, background subtraction and patterns merging) were further analyzed using either Irena SAS macros for Igor Pro 1 or SASfit program 2 .

Rheology measurements
Simple structural models based mainly on shape form factors of scattering objects are applied for SAXS analysis. For example, given that the X-ray scattering length densities of the blocks comprising the copolymer are similar, the SAXS patterns of the PEG 113 -PHPMA 100 spherical micelles can be fitted reasonably well by a relatively simple model based on a spherical form factor (see Figure 7f and the main text associated with this figure). This can be demonstrated by comparing results obtained from this model to results obtained from a more sophisticated micelle model. 3,4 Assuming that there is no penetration of the PEG coronal blocks in the PHPMA micelle core, the following expression for the spherical micelle form factor can be used 3, 4 : where R s is the radius of the micelle core and R g is the radius of gyration of the PEG , where x sol is the concentration of water within the core. The amplitude of the core self-term is Since the PEG corona contribution to the scattering signal is comparable to the scattering from the PHPMA core and Eq. (S4) is given by: . Due to a possible partial interpenetration of the coronal chains on adjacent micelles, R g (rather than 2R g ) is used in the expressions for the inter-micelle separation distances.
Size polydispersity of the micelles was determined assuming a normal distribution of the core radius (R s ).The spherical micelle model, Eq. (S4), produces a reasonably good fit to the SAXS data and mostly overlaps with the sphere model fitting ( Figure S6).
Programming tools for the Irena SAS Igor Pro macros were used for the model fitting, which yielded R s = 12.2 nm, R g = 1.8 nm, s = 3.3 nm and a = 3.1. It was also found that x sol = 0.50, suggesting a relatively high degree of hydration for the plasticized PHPMA chains. However, this observation is consistent with our earlier 1 H NMR spectroscopy studies. 6 A similarly high value was also observed for Pluronic-type block copolymers. 4 It was also found that single spherical micelles dominate the PEG 113 -PHPMA 100 aqueous dispersion (volume fraction ~ 0.9) with only a minor population of dimers and trimers (total volume fraction, k 2 + k 3 = 0.1). Fitting the SAXS pattern obtained after 1 second of X-ray exposure indicated that the proportion of dimers and trimers increased S10 significantly (k 2 + k 3 = 0.8). The core radius also slightly increased to 12.8 nm, presumably owing to thermal expansion and possible salvation.
If the micelle diameter is expressed as ) ( 2 g s R R + , the fitted sphere diameters (see Table   1)      Table S1. Characterization data obtained for various PEG 113 -PHPMA x diblock copolymer dispersions. These data were used to construct the detailed phase diagram shown in Figure 4 of the main text.