Using Dynamic Covalent Chemistry To Drive Morphological Transitions: Controlled Release of Encapsulated Nanoparticles from Block Copolymer Vesicles

Dynamic covalent chemistry is exploited to drive morphological order–order transitions to achieve the controlled release of a model payload (e.g., silica nanoparticles) encapsulated within block copolymer vesicles. More specifically, poly(glycerol monomethacrylate)–poly(2-hydroxypropyl methacrylate) (PGMA–PHPMA) diblock copolymer vesicles were prepared via aqueous polymerization-induced self-assembly in either the presence or absence of silica nanoparticles. Addition of 3-aminophenylboronic acid (APBA) to such vesicles results in specific binding of this reagent to some of the pendent cis-diol groups on the hydrophilic PGMA chains to form phenylboronate ester bonds in mildly alkaline aqueous solution (pH ∼ 10). This leads to a subtle increase in the effective volume fraction of this stabilizer block, which in turn causes a reduction in the packing parameter and hence induces a vesicle-to-worm (or vesicle-to-sphere) morphological transition. The evolution in copolymer morphology (and the associated sol–gel transitions) was monitored using dynamic light scattering, transmission electron microscopy, oscillatory rheology, and small-angle X-ray scattering. In contrast to the literature, in situ release of encapsulated silica nanoparticles is achieved via vesicle dissociation at room temperature; moreover, the rate of release can be fine-tuned by varying the solution pH and/or the APBA concentration. Furthermore, this strategy also works (i) for relatively thick-walled vesicles that do not normally exhibit stimulus-responsive behavior and (ii) in the presence of added salt. This novel molecular recognition strategy to trigger morphological transitions via dynamic covalent chemistry offers considerable scope for the design of new stimulus-responsive copolymer vesicles (and hydrogels) for targeted delivery and controlled release of cargoes. In particular, the conditions used in this new approach are relevant to liquid laundry formulations, whereby enzymes require protection to prevent their deactivation by bleach.

S2 immersed in an oil bath set at 70 °C to initiate the RAFT solution polymerization of GMA and stirred for 2 h at this temperature. The GMA polymerization was then quenched by exposure to air, followed by cooling the reaction mixture to room temperature. Ethanol (25 mL) was added to dilute the reaction solution, followed by precipitation into a ten-fold excess of dichloromethane in order to remove unreacted GMA monomer. The precipitate was isolated via filtration and washed with excess dichloromethane before being dissolved in methanol (50 mL). The crude polymer was precipitated for a second time by addition to excess dichloromethane and isolated by filtration. It was then dissolved in water and freezedried for 48 h to afford a pink power. The mean degree of polymerization of this PGMA macro-CTA was calculated based on 1 H NMR spectrum (see Figure S1). macro-CTA is as follows: PGMA 45 macro-CTA (0.12 g, 16 µmol), HPMA monomer (0.39 g, 2.72 mmol), and ACVA (1.10 mg, 4.0 µmol; macro-CTA/ACVA molar ratio = 4.0) were added into a 25 mL round-bottomed flask, prior to addition of water to produce a 15% w/w solution. This reaction solution was purged with nitrogen gas for 30 min at 20 °C prior to immersion into an oil bath set at 70 °C. The reaction mixture was stirred for 3 h to ensure essentially complete conversion of the HPMA monomer, 1 then the polymerization was quenched by exposure to air, followed by cooling to ambient temperature. For the closely related synthesis of PGMA 45 -PHPMA 240 and PGMA 45 -PHPMA 300 , the mass of added HPMA monomer was 0.56 g (3.84 mmol) and 0.70 g (4.80 mmol), respectively, and the volume of water was also adjusted accordingly in order to maintain a constant 15% w/w solids.

Synthesis of PGMA
Morphological transitions for PGMA 45 -PHPMA x diblock copolymer vesicles induced by dynamic covalent chemistry. The initial 15% w/w aqueous vesicle dispersion was diluted to S3 1.0% w/w using water and then adjusted to pH 10.5 by addition of 0.02 M NaOH solution.
APBA was dissolved in either 1 M or 0.1 M NaOH solution to produce a 0.60% w/w APBA aqueous solution at pH 10.5. This solution was stored in the dark prior to use. 0.40 g of the 1.0% w/w aqueous vesicle dispersion was then mixed with the APBA solution at the desired volumetric ratio in a 10 mL vial, and the resulting mixture was further diluted using NaOH solution or water produce a 0.20% w/w aqueous vesicle dispersion at pH 10.5. The sealed vial was stored at room temperature in the dark and aged for the desired time period for turbidimetry, TEM and DLS studies.

Synthesis of silica nanoparticles loaded PGMA 58 -PHPMA 250 diblock copolymer vesicles.
The details for the preparation of PGMA 58 -PHPMA 250 diblock copolymer vesicles in the presence or absence of an aqueous dispersion of 20 nm diameter glycerol-functional silica nanoparticles were recently reported by Mable et al. 2 Release of silica nanoparticles from PGMA 58 -PHPMA 250 diblock copolymer vesicles.
The silica nanoparticle release experiments were similar to those described for the PGMA 45 -PHPMA x (where x = 170 or 240) diblock copolymer vesicles, except that NaOH was replaced by aqueous ammonia in all cases. The loaded silica content was 8% w/w as calculated via thermogravimetric analysis ( Figure S8). Characterization NMR Spectroscopy. 1 H NMR spectra were recorded in CD 3 OD using a 400 MHz Bruker Avance-500 spectrometer (64 scans averaged per spectrum). 11 B NMR spectra were recorded in deionized water containing 10% D 2 O at the desired pH using quartz NMR tubes on a 500 MHz Bruker Avance III HD spectrometer operating at 160.46 MHz (typical number of scans per spectrum = 256).

Gel Permeation Chromatography (GPC).
Polymer molecular weights and dispersities were determined using a DMF GPC set-up operating at 60 °C and comprising two Polymer

Transmission Electron Microscopy (TEM). Copper TEM grids (Agar Scientific, UK) were
surface-coated in-house to yield a thin film of amorphous carbon. The grids were then plasma glow-discharged for 30 s to create a hydrophilic surface. Each dispersion (0.20% w/w, 5 µL) was placed on such a grid for 30 s and then blotted with filter paper to remove excess solution. To stain the aggregates, a 5 µL drop of 0.75% w/w uranyl formate solution was placed on the sample-loaded grid for 60 s and then carefully blotted to remove excess stain.
The grids were then dried using a vacuum hose. Imaging was performed at 80 kV using a FEI Tecnai Spirit microscope equipped with a Gatan 1kMS600CW CCD camera.
Turbidimetry Studies. Absorbance spectra were recorded at 20 °C every 10 min over 15 h using a Shimadzu UV-1800 spectrometer operating at a fixed wavelength of 450 nm.

S5
Rheology. Storage (G′) and loss (G″) moduli were determined at 20 °C for a 10% w/w aqueous dispersion of PGMA 45 −PHPMA 170 vesicles at pH 10.5 using a TA Instruments AR-G2 rheometer. A cone-and-plate geometry (40 mm 2° aluminum cone) was used for these measurements, which were conducted at a fixed strain of 1.0% and an angular frequency of 1.0 rad s −1 .

Small-angle X-ray scattering (SAXS)
SAXS patterns were recorded at a synchrotron facility (ESRF, station ID02, Grenoble, France) using monochromatic X-ray radiation (wavelength λ = 0.0995 nm; sample-todetector distance = 5.0042 m; q ranged from 0.015 to 1.5 nm -1 , where q = 4π sin θ/λ is the length of the scattering vector and θ is one-half of the scattering angle) and a Ravonix MX-170HS CCD detector. A glass capillary of 2.0 mm diameter was used as a sample holder.
Scattering data were reduced using standard routines provided by beamline personnel and were further analyzed using Irena SAS macros for Igor Pro. 3 Water was used for the absolute intensity calibration. Time-resolved measurements were conducted on a 10% w/w aqueous dispersion of PGMA 45 -PHPMA 170 vesicles at pH 10.5 immediately after adding ABPA.
Thermogravimetric analysis (TGA). Analyses were conducted on freeze-dried samples that were heated in air to 800 °C at a heating rate of 15 °C min -1 using a TA Instruments Q500 instrument. Figure S1. 1    Evolution of the apparent sphere-equivalent DLS average diameter recorded for PGMA 58 -PHPMA 250 diblock copolymer nano-objects with ageing time in the absence and presence of 11.2 mM APBA at pH 10.5. S11 Figure S8. Thermogravimetric analysis curve recorded for silica-loaded PGMA 58 -PHPMA 250 diblock copolymer vesicles after centrifugation to remove the excess silica nanoparticles. The residual mass observed at 600 °C is attributed to the incombustible encapsulated silica nanoparticles within the vesicles.