Anionic block copolymer vesicles act as Trojan horses to enable efficient occlusion of guest species into host calcite crystals

‘Trojan Horse’ anionic poly(methacrylic acid)–poly(benzyl methacrylate) vesicles enable efficient incorporation of either nanoparticles or soluble small molecules within calcite.

S2 macro-CTA was dialyzed first against a 9:1 water/methanol mixture and then against deionized water. The purified polymer was isolated by lyophilization. A mean DP of 69 was calculated for this macro-CTA using 1 H NMR spectroscopy by comparing the integrated signal intensity assigned to the aromatic protons at 7.2-7.4 ppm with that due to the methacrylic backbone at 0.4-2.5 ppm. After exhaustive methylation using excess trimethylsilyldiazomethane, THF GPC analysis of this PMAA 69 macro-CTA indicated an M n of 9 700 g mol -1 and an M w /M n of 1.16.

Synthesis of poly(methacrylic acid) 62 (PMAA 62 ) macro-CTA
PMAA 62 macro-CTA was prepared using a commercially-available RAFT CTA (CPCP). sealed reaction vessel was purged with nitrogen and placed in a pre-heated oil bath at 70°C for 3 h. The resulting PMAA macro-CTA was dialyzed first against a 9:1 water/methanol mixture and then against deionized water. The purified polymer was isolated by lyophilization. A mean DP of 62 was calculated for this macro-CTA using 1 H NMR spectroscopy by comparing the integrated signal intensity assigned to the aromatic protons at 7.3-8.0 ppm with that due to the methacrylic backbone at 0.4-2.5 ppm. After exhaustive methylation using excess trimethylsilyldiazomethane, THF GPC analysis indicated an M n of 8 000 g mol -1 and an M w /M n of 1.16.

Synthesis of poly(glycerol monomethacrylate) 51 (PGMA 51 ) macro-CTA
CPDB RAFT agent (0.46 g, 2.0 mmol, ~ 80% purity), ACVA initiator (0.117 g, 0.40 mmol, [CPDB]/[ACVA] = 5.0) and anhydrous ethanol (30.0 g) were added to a round-bottomed S3 flask containing a magnetic stirrer bar. Once the CPDB and ACVA were fully dissolved, GMA monomer (20.0 g, 0.125 mol) was added to target a degree of polymerization (DP) of 60. This pink solution was sparged with N 2 for 30 min, before being placed into a pre-heated oil bath set at 70 °C. The GMA polymerization was quenched after 2 h by cooling the flask in ice, followed by exposure to air. The crude polymer was purified twice by precipitating into a ten-fold excess of dichloromethane. The precipitate was redissolved in water and the final macro-CTA was obtained in powdered form by lyophilization. 1 H NMR spectroscopy indicated a DP of 51 for this PGMA macro-CTA by comparing the integrated signals assigned to the five methacrylic backbone protons (0.4 to 2.5 ppm) with the integrated aromatic signals corresponding to the five protons associated with the RAFT CTA end-group.
DMF GPC analysis (vs. poly(methyl methacrylate) standards) indicated M n and M w /M n values of 12 900 g mol -1 and 1.18, respectively.

Precipitation of calcite crystals in the presence of cargo-loaded vesicles
An aqueous solution (10 mL) comprising CaCl 2 (1.5 mM) and 0.10 % w/w vesicles was placed in a dessicator. CaCO 3 crystals were precipitated onto a glass slide placed at the base of this aqueous solution by exposure to ammonium carbonate vapor (2-3 g, placed at the bottom of the dessicator) for 24 h at 20 ºC. Then the glass slide was removed from the solution and washed three times with deionized water followed by three rinses with ethanol.
Each occlusion experiment was repeated at least twice and consistent results were obtained in each case. 1 H NMR spectra were recorded using a Bruker Avance 400 spectrometer operating at 400 MHz using either CD 3 OD or d 8 -THF solvents.

Gel permeation chromatography (GPC)
For THF GPC studies, the carboxylic acid groups on the PMAA macro-CTA were methylated using trimethylsilyldiazomethane, as reported by Couvreur et al. 3 The GPC set-up consisted of two 5 μM Mixed C columns connected to a WellChrom K-2301 refractive index detector. The mobile phase was HPLC-grade THF containing 1.0% glacial acetic acid and S6 0.05% w/v butylhydroxytoluene (BHT) at a flow rate of 1.0 mL min -1 . Molecular weights were calculated with respect to a series of near-monodisperse poly(methyl methacrylate) standards.
The DMF GPC set-up was operated at 60 °C with the instrument comprising two Polymer Laboratories PL gel 5 µm Mixed C columns and one PL polar gel 5 µm guard column connected in series to a Varian 390-LC multi-detector suite (refractive index detector only) and a Varian 290-LC pump injection module. The GPC eluent was HPLC-grade DMF containing 10 mM LiBr and was filtered prior to use. The flow rate was 1.0 mL min -1 and DMSO was used as a flow-rate marker. Calibration was conducted using a series of ten nearmonodisperse poly(methyl methacrylate) standards (M n = 6.25 × 10 2 -6.18 × 10 5 g mol -1 , K = 2.094 × 10 -3 , α = 0.642). Chromatograms were analyzed using Varian Cirrus GPC software.

Dynamic light scattering (DLS)
DLS measurements were conducted using a Malvern Zetasizer NanoZS instrument by detecting back-scattered light at an angle of 173°. Aqueous dispersions of the samples were diluted to 0.1 % w/w using deionized water. Hydrodynamic diameters were calculated using the Stokes-Einstein equation.

Optical microscopy
Optical microscopy images were recorded using a Motic DMBA300 digital biological microscope equipped with a built-in camera and analyzed using Motic Images Plus 2.0 ML software.

Transmission electron microscopy (TEM)
TEM images were obtained by depositing 0.15 % w/v alcoholic vesicle dispersions (or an aqueous vesicle dispersion in the presence of 6 mM CaCl 2 ) onto palladium-copper grids (Agar Scientific, UK) coated with carbon film. The grids were treated with a plasma glow discharge for approximately 30 seconds to create a hydrophilic surface prior to addition of a 5 µL droplet of the dispersion using a micropipet. Excess solvent was removed via blotting and the grid was stained with uranyl formate for 30 seconds. Excess stain was removed via blotting and the grid was carefully dried under vacuum. Imaging was performed using a FEI Tecnai G2 Spirit instrument.

Scanning electron microscopy (SEM)
Samples were fractured by placing a clean glass slide on top of the calcite-coated glass slide, pressing down lightly and twisting one slide relative to the other. The resulting randomlyfractured calcite crystals were examined by scanning electron microscopy using either an Inspect F instrument after sputter-coating with gold (15 mA, 1.5 min) or imaged directly using a Nova NanoSEM 450 instrument without any sputter-coating. A relatively low accelerating voltage (2-5 kV) was applied in order to prevent sample charging. To image the vesicles, a droplet of an aqueous dispersion of vesicles was dried onto a clean glass slide and then coated with gold prior to imaging. Sample milling was performed using a FEI Helio G4 CX dual beam-high resolution monochromated FEG SEM instrument equipped with a focused ion beam (FIB). The operating voltage was 30 kV and the beam current was varied between 0.1 and 5 nA.

Small-angle X-ray scattering (SAXS)
SAXS patterns were collected at an international synchrotron source (ESRF, station ID02, Grenoble, France) using monochromatic X-ray radiation (λ = 0.0995 nm, with q ranging from 0.013 to 1.8 nm -1 , where q is the length of the scattering vector, i.e. q = 4π sin θ/λ and θ is one-half of the scattering angle) and a Ravonix MX-170HS CCD detector. Glass capillaries of 2.0 mm diameter were used as sample holders. Scattering data were reduced using standard software packages available at the beamline and were further analyzed using Irena SAS macros for Igor Pro. 4 Water was used for the absolute intensity calibration.

Other measurements
Fluorescence microscopy images were recorded on a Zeiss Axio Scope A1 microscope fitted with an AxioCam 1Cm1 monochrome camera. Images were captured and processed using ZEN lite 2012 software. Confocal fluorescence images were recorded using a Nikon A1 microscope equipped with Nikon elements software. Raman spectra were recorded using a Renishaw 2000 Raman microscope equipped with a 785 nm diode laser. Thermogravimetric analysis (TGA) was conducted from 20 °C to 900 °C in air using a Perkin-Elmer Pyris 1 instrument at 10 °C min -1 .      The vesicles shown in Fig. S7a and S7b were not stained, so their vesicular morphology is not readily discernible. However, uranyl formate staining of the same samples clearly revealed a well-defined vesicular morphology in each case (see Fig. 1). Indeed, this morphology was confirmed by the gradient of -2 observed in the low q range of each SAXS pattern shown in Fig. S7c. 5 Hence the silica mass % within these vesicles can be calculated as follows: Assuming that the relative mass contents % for the PMAA 69 -PBzMA 200 vesicles, silica, CaO and CO 2 are x, y, m, and n, respectively, then the following four equations can be obtained: 99.2% × x + n = 48.7 0.8% × x + 97.3% × y = 11.6% × (x + y) Solving the above equations, x = 9.4, y = 1.2 and m = 50.1, n = 39.3 So, the extent of vesicle occlusion within the calcite is 9.4% by mass.

S19
The cartoon below shows the outer radius (R) and inner radius (r) of the vesicles. The mean vesicle diameter (2R) can be estimated by analyzing SEM images and the vesicle membrane thickness was calculated by TEM analysis. For the silica-loaded PMAA 69 -PBzMA 200 vesicles, the vesicle diameter = 208 ± 46 nm and the membrane thickness (T) = 19 ± 3 nm.
Then the effective density of empty vesicles, , can be calculated as follows.
The solid-state density of the dried PMAA 69 -PBzMA 200 vesicles was determined to     Based on Fig. S14a, the fluorescein appears to merely adsorb onto the surface of the calcite crystals. It is not possible to tell whether any fluorescein is incorporated within the crystals from such images. However, TGA analysis suggests that the the extent of fluorescein occlusion must be negligible. In contrast, for calcite prepared in the presence of 0.1% w/w fluorescein-loaded vesicles, the whole crystal is highly fluorescent, indicating the uniform