Occlusion of Sulfate-Based Diblock Copolymer Nanoparticles within Calcite: Effect of Varying the Surface Density of Anionic Stabilizer Chains

Polymerization-induced self-assembly (PISA) offers a highly versatile and efficient route to a wide range of organic nanoparticles. In this article, we demonstrate for the first time that poly(ammonium 2-sulfatoethyl methacrylate)-poly(benzyl methacrylate) [PSEM–PBzMA] diblock copolymer nanoparticles can be prepared with either a high or low PSEM stabilizer surface density using either RAFT dispersion polymerization in a 2:1 v/v ethanol/water mixture or RAFT aqueous emulsion polymerization, respectively. We then use these model nanoparticles to gain new insight into a key topic in materials chemistry: the occlusion of organic additives into inorganic crystals. Substantial differences are observed for the extent of occlusion of these two types of anionic nanoparticles into calcite (CaCO3), which serves as a suitable model host crystal. A low PSEM stabilizer surface density leads to uniform nanoparticle occlusion within calcite at up to 7.5% w/w (16% v/v), while minimal occlusion occurs when using nanoparticles with a high PSEM stabilizer surface density. This counter-intuitive observation suggests that an optimum anionic surface density is required for efficient occlusion, which provides a hitherto unexpected design rule for the incorporation of nanoparticles within crystals.

S3 prepared by RAFT dispersion polymerization, the ethanol co-solvent was removed by dialysis against deionized water (dialysis tubing MWCO = 5,000).

Synthesis of PGMA 70 (G 70 ) macro-CTA via RAFT solution polymerization
A typical protocol for the synthesis of PGMA 70 macro-CTA is as follows. To a roundbottomed flask containing CPCP RAFT agent (0.96 g, 3.43 mmol), GMA monomer (38.44 g, 0.24 mol) and anhydrous ethanol (59.40 g, 1.28 mol) were added to afford a target degree of polymerization (DP) of 70. To this mixture, ACVA initiator (0.19 g, 0.69 mmol, CTA/ACVA molar ratio = 5.0) was added and the resulting pink solution was sparged with N 2 for 20 min, before the sealed flask was immersed into an oil bath set at 70 °C. After 2.5 h (88 % conversion as judged by 1 H NMR) the polymerization was quenched by immersing the reaction flask in an ice bath and exposing its contents to air. The polymer solution was then precipitated into a ten-fold excess of dichloromethane and washed three times in this solvent before being placed under high vacuum for three days at 40 °C. 1
The vial was sealed and purged with N 2 for 30 min prior to transfer to a preheated oil bath set at 70 °C for 24 h.

Precipitation of calcium carbonate crystals in the presence of nanoparticles
CaCO 3 crystals were precipitated onto a glass slide placed at the base of an aqueous solution containing 1.5 mM CaCl 2 and various diblock copolymer nanoparticles ranging in concentration from 0.001 % w/w to 0.1 % w/w by exposure to ammonium carbonate vapor for 24 h at 20 º C. 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.

Precipitation of zinc oxide crystals in the presence of nanoparticles
Aqueous copolymer nanoparticle dispersions (0.20 mL, 5.0 % w/w) were added to a twonecked flask containing an aqueous solution of zinc nitrate hexahydrate (0.446 g, 1.50 mmol) to give a total volume of 98.0 mL. This flask was connected to a condenser and transferred to a preheated oil bath set at 90 °C and the reaction mixture was magnetically stirred to achieve thermal equilibrium (typically 30 min). ZnO formation occurred on addition of 2.0 mL of an aqueous solution of HMTA (0.210 g, 1.50 mmol). The reaction was quenched after 90 min by cooling in an ice-water bath. The resulting nanocomposite crystals were isolated by centrifugation and washed several times using water or ethanol, followed by drying under vacuum at 40 °C. 1 H NMR spectra were recorded using a Bruker Avance 400 spectrometer operating at 400 MHz using D 2 O, CD 3 OD or d 6 -DMSO as solvents.

Gel permeation chromatography (GPC)
Aqueous GPC analysis was performed using an Agilent Technologies Infinity 1260 set-up equipped with two PL aquagel-OH 30 8 µm columns running at 35 °C. The GPC eluent comprised deionized water containing 30 vol % methanol at pH 9 at a flow rate of 1.0 mL min -1 . Calibration was achieved using a series of near-monodisperse poly(ethylene oxide) standards ranging from 4.1 x 10 3 to 6.92 x 10 5 g mol -1 . 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.
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 copolymer nanoparticles were diluted to 0.15 % w/v using deionized water. Aqueous electrophoresis measurements were conducted using disposable folded capillary cells supplied by Malvern (DTS1070) using the same instrument using 1 mM NaCl as a background electrolyte. For zeta potential vs. Ca 2+ concentration studies, the concentration of diblock copolymer S6 nanoparticle was fixed at 0.01 % w/w. Each measurement was repeated three times and averaged to give the mean zeta potential.

Transmission electron microscopy (TEM)
TEM images were obtained by depositing 0.15 % w/v aqueous dispersion of copolymer nanoparticles 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 the aqueous nanoparticle dispersion (5 µL). 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.

Field emission-scanning electron microscopy (FE-SEM)
Calcite crystal morphologies were examined using a high-resolution field emission-scanning electron microscope (Nova NanoSEM 450). Glass slides supporting the CaCO 3 crystals were mounted on stubs using adhesive conductive pads with no further coating. Samples were fractured by placing a clean glass slide on top of the glass slide supporting the calcite crystals, pressing down lightly and twisting one slide relative to the other. A relatively low accelerating voltage (2-3 kV) was applied in order to prevent sample charging.

X-ray photoelectron spectroscopy (XPS)
XPS samples were prepared by placing a droplet of an aqueous dispersion of diblock copolymer nanoparticles onto clean indium foil and allowing evaporation to occur overnight at 20 °C. Powder samples were directly pressed onto clean indium foil. XPS data were acquired using a Kratos Axis Ultra DLD instrument equipped with monochromatic Al X-ray radiation at 6.0 mA and 15 kV at a typical base pressure of 10 -8 Torr. The step size was 0.5 eV for the survey spectra (pass energy = 160 eV) and 0.05 eV for the high resolution spectra (pass energy = 20 eV). Raw data were corrected using a transmission function characteristic S7 of the instrument, determined using software provided by the National Physical Laboratory.
The calibrated spectra can then be quantified using theoretically-derived Scofield relative sensitivity factors.

Other measurements
Optical microscopy images were recorded using a Motic DMBA300 digital biological  Figure S13). The amount of polymer-bound calcium was calculated from the total amount of Ca 2+ added and the amount of free Ca 2+ measured. FT-IR spectra were recorded on KBr pellets using a Nicolet 7199 FT-IR spectrometer. UV-visible spectra were recorded at 20 °C using a PerkinElmer Lambda 25 instrument operating between 200 and 800 nm.

Extent of occlusion determined by thermogravimetric analysis
Heating calcite control led to decomposition above 625 °C leaving a final oxide residue of 56.4 % w/w, which is slight higher than theoretical value (56.0 % w/w), most probably due to instrumental error. TGA studies indicated that PSEM 73 -PBzMA 300 (emulsion)/calcite nanocomposite crystal exhibited a 3.7 % weight loss between 250 °C and 600 °C. This is due to pyrolysis of copolymer nanoparticles located on or near the outer surfaces of the crystals.

S8
The additional 44.1 % weight loss observed between 600 °C and 850 °C is due to both copolymer pyrolysis and CO 2 evolution from the thermal decomposition of CaCO 3 . As the PSEM 73 -PBzMA 300 copolymer nanoparticles were fully pyrolysed, all of the 52.2 % w/w residue is CaO derived from the decomposition of CaCO 3 , corresponding to 40.3 % w/w CO 2 .
Thus the copolymer nanoparticle content of the original nanocomposite crystals can be calculated to be 7.   Well-defined S 73 -B 100 (emulsion) cannot be prepared as the core-forming block is too short].
S21 Figure S12. DLS particle size distributions recorded for PSEM 73 -PBzMA 300 (dispersion) copolymer nanoparticles before and after dialysis against water (52 nm before dialysis vs. 60 nm after dialysis). The slightly higher DLS diameter observed in pure water simply reflects slightly greater stretching of the anionic stabilizer chains. Using SAXS and 1 H NMR spectroscopy, we have recently shown that both ethanol and water are non-solvents for the PBzMA core-forming block. 8 Thus dialysis from 2:1 ethanol/water into pure water is not expected to change the dimensions of these PSEM 73 -PBzMA 300 nanoparticles.
S22 Figure S13. Linear calibration curve (R 2 > 0.999; y = 25.41x + 452.3) obtained using a calcium-selective electrode for the determination of calcium complexation by S x -B y diblock copolymer nanoparticles. The amount of copolymer-bound calcium was calculated from the difference between the initial Ca 2+ concentration and the concentration of free Ca 2+ measured using the ion-selective electrode. comparable with that of the S 73 -B 300 (emulsion) copolymer nanoparticles (see Table 1). These copolymer nanoparticle/ZnO nanocomposite crystals were heated up to 900 °C in air to remove the copolymer component via pyrolysis and then gold-coated prior to SEM imaging.