Synthesis of poly(stearyl methacrylate)-poly(2-hydroxypropyl methacrylate) diblock copolymer nanoparticles via RAFT dispersion polymerization of 2-hydroxypropyl methacrylate in mineral oil

Csilla György, Saul J. Hunter, Chloé Girou, Matthew J. Derry and Steven P. Armes*
Dainton Building, Department of Chemistry, The University of Sheffield, Brook Hill, Sheffield, South Yorkshire S3 7HF, UK. E-mail: s.p.armes@shef.ac.uk

Received 17th April 2020 , Accepted 8th June 2020

First published on 16th June 2020


Poly(stearyl methacrylate)-poly(2-hydroxypropyl methacrylate) (PSMA-PHPMA) diblock copolymer nanoparticles are synthesized via reversible addition–fragmentation chain transfer (RAFT) dispersion polymerization of 2-hydroxypropyl methacrylate (HPMA) in mineral oil at 90 °C. The relatively short PSMA precursor (mean degree of polymerization = 9) remains soluble in mineral oil, whereas the growing PHPMA block quickly becomes insoluble, resulting in polymerization-induced self-assembly (PISA). Relatively high HPMA monomer conversions (≥98%) were achieved within 70 min as confirmed by in situ 1H NMR spectroscopy studies, while gel permeation chromatography (GPC) analyses indicated high blocking efficiencies and relatively narrow molecular weight distributions (Mw/Mn ≤ 1.37) for all PISA syntheses. Depending on the precise synthesis conditions, this PISA formulation can produce diblock copolymer spheres, worms or vesicles; a pseudo-phase diagram has been constructed to enable reproducible targeting of each pure phase. Thus this is a rare example of the use of a commercially available polar monomer for PISA syntheses in non-polar media that offers access to the full range of copolymer morphologies. The resulting nanoparticles were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), oscillatory rheology and small-angle X-ray scattering (SAXS). Interestingly, PSMA9-PHPMA70 worms undergo an unusual (partial) worm-to-vesicle transition at elevated temperature. Finally, PSMA9-PHPMA50 spheres were evaluated as putative Pickering emulsifiers. Using lower water volume fractions produced water-in-oil (w/o) emulsions after high shear homogenization, as expected. However, using higher water volume fractions, shear rates or copolymer concentrations favored the formation of w/o/w Pickering double emulsions.


Introduction

It is well-known that AB diblock copolymers undergo self-assembly in a selective solvent.1,2 This provides access to a range of diblock copolymer morphologies, with spheres, worms and vesicles being the most common.3–5 The preferred copolymer morphology is usually dictated by the relative volume fractions of the two blocks, as indicated by the geometric packing parameter.6 However, such diblock copolymer nano-objects are typically generated in highly dilute solution (<1%) using various post-polymerization processing techniques, such as a solvent switch,4 a pH switch7 or thin film rehydration.8 This is a significant limitation that has hitherto hindered industrial scale-up for potential applications.

In principle, polymerization-induced self-assembly (PISA)9–12 offers a highly convenient route to diblock copolymer nano-objects at up to 50% w/w solids. PISA utilizes controlled living radical polymerization techniques such as reversible addition–fragmentation chain transfer (RAFT) polymerization.13–15 Selecting a suitable solvent for the respective monomer building blocks is critical for a successful PISA formulation: the precursor ‘A’ block requires a good solvent to act as an effective steric stabilizer while the growing ‘B’ block requires a non-solvent. Initially, the diblock copolymer chains remain fully soluble, because unreacted monomer acts as a co-solvent. However, a critical degree of polymerization is eventually attained whereby the ‘B’ block becomes insoluble, driving in situ self-assembly to produce a colloidal dispersion of sterically-stabilized nanoparticles.

The design rules for PISA are generic: such syntheses can be conducted in a wide range of solvents, including water, lower alcohols and mixtures thereof.11,16–21 Recently, we have studied RAFT dispersion polymerization in various non-polar media such as n-alkanes,22–26 poly(α-olefins),12 mineral oil12,27–30 or low-viscosity silicone oil.31 Potential applications include next-generation nanoparticle lubricants for automotive engine oils,30 a new high-temperature oil-thickening mechanism27 and novel viscosity modifiers for silicone oils.31

Poly(2-hydroxypropyl methacrylate) (PHPMA) has been utilized for a wide range of PISA formulations.32–42 For example, Blanazs et al. studied the evolution of copolymer morphology during the RAFT aqueous dispersion polymerization of HPMA, which provided important mechanistic insights regarding the worm-to-vesicle transition.32 In closely-related work, using PHPMA as a weakly hydrophobic structure-directing block enables the rational design of thermoresponsive worms33,35,36 and vesicles.34 Moreover, utilizing PHPMA in conjunction with highly biocompatible steric stabilizer blocks such as poly(glycerol monomethacrylate) [PGMA]43 or poly(ethylene glycol) [PEG]37,44 enable the soft hydrogels formed by semi-concentrated worm dispersions45 to be evaluated as novel cell storage media.44,46–48 Zehm and co-workers demonstrated that PHPMA can also be used as a steric stabilizer block for the RAFT alcoholic dispersion polymerization of benzyl methacrylate.42 Rymaruk et al. briefly reported that PHPMA could serve as a core-forming block when exploring RAFT dispersion polymerization formulations in silicone oil.31 However, only kinetically-trapped spheres could be obtained in this latter case. As far as we are aware, the current study is the first to report the use of PHPMA as a structure-directing block to access spheres, worms or vesicles via RAFT-mediated PISA in non-polar media.

Pickering emulsions are particle-stabilized emulsions and have been recognized for more than a century.49,50 It is well-known that the type of emulsion obtained depends on the particle contact angle, which is in turn dictated by the nanoparticle wettability.51 Since 2012, various types of diblock copolymer nano-objects have been evaluated as Pickering emulsifiers.52–55 For example, hydrophilic diblock copolymer nanoparticles prepared via aqueous PISA enable the formation of oil-in-water (o/w) Pickering emulsions.53–55 In contrast, hydrophobic nanoparticles prepared in non-polar solvents such as n-alkanes or mineral oil favour the formation of water-in-oil (w/o) emulsions.56,57 Interestingly, Thompson et al. utilized hydrophobic and hydrophilic worms in turn to prepare water-in-oil-in-water (w/o/w) Pickering emulsions.58

Herein, we report the synthesis of PSMA9-PHPMAx diblock copolymer nano-objects via RAFT dispersion polymerization of HPMA in mineral oil at 90 °C. Using a relatively short PSMA9 precursor block ensures access to spheres, worms and vesicles.28 A pseudo-phase diagram has been constructed for a series of PSMA9-PHPMAx nanoparticles by varying the copolymer concentration from 15% to 30% w/w, confirming that a pure worm phase can be obtained over a relatively narrow range of diblock copolymer compositions (x = 67 to 70). The thermoresponsive nature of such worms is briefly explored and PSMA9-PHPMA50 spheres are evaluated as putative Pickering emulsifiers.

Experimental

Materials

2-Hydroxypropyl methacrylate (HPMA) was donated by GEO Specialty Chemicals (UK) and used without further purification. Stearyl methacrylate (SMA) was purchased from Santa Cruz Biotechnology Ltd (USA) and was used without further purification. Benzyl methacrylate (BzMA, 96%) was purchased from Sigma Aldrich, passed through an inhibitor remover column to remove monomethyl ether hydroquinone (MMEHQ) and then stored at −20 °C prior to use. 2-Cyano-2-propyl dithiobenzoate (CPDB), CDCl3 and n-dodecane were purchased from Sigma Aldrich (UK). 2,2′-Azoisobutyronitrile (AIBN) was obtained from Molekula (UK). tert-Butyl peroxy-2-ethylhexanoate (T21s) was purchased from AkzoNobel (The Netherlands). CD2Cl2 was purchased from Goss Scientific (UK). API Group III mineral oil (viscosity = 3.1 cSt at 100 °C) was kindly provided by The Lubrizol Corporation Ltd (Hazelwood, Derbyshire, UK). All other solvents were purchased from Fisher Scientific (UK) and were used as received.

Synthesis of poly(stearyl methacrylate) (PSMA9) precursor via RAFT solution polymerization

The synthesis of the PSMA9 homopolymer precursor was conducted at 50% w/w solids as follows. SMA (34.0 g; 100.4 mmol), CPDB (4.40 g; 19.9 mmol; target degree of polymerization = 5), AIBN (659 mg; 4.01 mmol; CPDB/AIBN molar ratio = 5.0) and toluene (39 g) were weighed into a 250 mL round-bottomed flask. The sealed reaction vessel was purged with nitrogen for 30 min, then placed in a pre-heated oil bath at 70 °C with stirring for 4 h. The SMA polymerization was quenched by exposing the reaction solution to air and cooling to room temperature. A final SMA conversion of 77% was determined by 1H NMR spectroscopy. The crude homopolymer was purified by two consecutive precipitations into a ten-fold excess of ethanol. The mean degree of polymerization (DP) of this macro-CTA was calculated to be 9 using 1H NMR spectroscopy by comparing the integrated aromatic protons from the dithiobenzoate end-group at 6.8–8.0 ppm to the two oxymethylene protons assigned to the SMA residues at 3.6–4.0 ppm. THF GPC analysis using a refractive index detector and a series of near-monodisperse poly(methyl methacrylate) standards indicated an Mn of 4500 g mol−1 and an Mw/Mn of 1.12.

Synthesis of poly(stearyl methacrylate)-poly(2-hydroxypropyl methacrylate) (PSMA9-PHPMAx) diblock copolymer nanoparticles via RAFT dispersion polymerization of HPMA in mineral oil

The protocol for the synthesis of PSMA9-PHPMA150 diblock copolymer vesicles at 15% w/w solids is representative and was conducted as follows. PSMA9 (0.040 g; 12.2 μmol), HPMA (0.26 g; 1.84 mmol), T21s initiator (0.53 mg; 2.45 μmol; 10.0% v/v in mineral oil; PSMA9/T21s molar ratio = 5.0) and mineral oil (1.73 g) were weighed into a vial and this reaction mixture was purged with nitrogen gas for 30 min. The sample vial was then immersed into a pre-heated oil bath at 90 °C and the reaction mixture was magnetically stirred for 5 h. 1H NMR spectroscopy studies indicated more than 98% HPMA monomer conversion (the integrated vinyl proton signals at 5.0–6.0 ppm were compared with the integrated polymer backbone signals at 3.5–4.0 ppm). THF GPC analysis indicated an Mn of 18[thin space (1/6-em)]800 g mol−1 and an Mw/Mn of 1.37. Other diblock copolymer compositions were targeted at 15% to 30% w/w solids by using the same mass of PSMA9 and adjusting the HPMA/PSMA9 molar ratio and the volume of mineral oil accordingly.

Preparation of water-in-oil Pickering emulsions

Water (0.50 mL, volume fraction = 0.25) was homogenized in turn with an 0.03–1.0% w/w PSMA9-PHPMA50 diblock copolymer dispersion in mineral oil (1.50 mL) for 2 min at 20 °C using an IKA Ultra-Turrax T-18 homogenizer equipped with a 10 mm dispersing tool operating at 13[thin space (1/6-em)]500 rpm. After appropriate dilution, the resulting aqueous emulsion droplets were imaged by optical and fluorescence microscopy and the volume-average droplet diameter was determined by laser diffraction. Other w/o Pickering emulsions were also prepared at water volume fractions of 0.125 and 0.375.

Preparation of water-in-oil-in-water Pickering double emulsions

Water (1.0 mL, volume fraction = 0.50) was homogenized in turn with an 1.0% w/w PSMA9-PHPMA50 diblock copolymer dispersion in mineral oil (1.0 mL) for 2 min at 20 °C using an IKA Ultra-Turrax T-18 homogenizer equipped with a 10 mm dispersing tool. The shear rate was systematically varied between 3500 rpm and 24[thin space (1/6-em)]000 rpm. After appropriate dilution, the resulting w/o/w double emulsions were visualized by optical microscopy and fluorescence microscopy. Other w/o/w Pickering emulsions were also prepared at water volume fractions of 0.75 and 0.875.

1H NMR spectroscopy

1H NMR spectra were recorded in either CD2Cl2 or CDCl3 using a 400 MHz Bruker Avance III spectrometer. Typically 64 scans were averaged per spectrum. In situ 1H NMR spectra were recorded using the same spectrometer in order to study the HPMA polymerization kinetics during the synthesis of PSMA9-PHPMA150 vesicles at 15% w/w solids in mineral oil and also PSMA9-PBzMA150 vesicles at 18% w/w solids in the same solvent. A 0.20 mL aliquot of the reaction mixture was transferred into an NMR tube equipped with an inert J-Young's tap under a nitrogen atmosphere. A capillary tube containing 0.28 M toluene dissolved in d6-DMSO was flame-sealed and used as an external standard (and also a solvent lock). A reference spectrum was recorded at 20 °C prior to heating the reaction mixture up to 90 °C. Spectra were recorded approximately every 2 min for the first 20 min and approximately every 6 min thereafter. Spectra were acquired in eight transients using a 30° excitation pulse and a delay time of 5 s over a spectral window of 16 kHz with 64 k data points.

Gel permeation chromatography (GPC)

Molecular weight distributions (MWDs) were assessed by GPC using THF eluent. The THF GPC system was equipped with two 5 μm (30 cm) mixed C columns and a WellChrom K-2301 refractive index detector operating at 950 ± 30 nm. The THF mobile phase contained 2.0% v/v triethylamine and 0.05% w/v butylhydroxytoluene (BHT) and the flow rate was fixed at 1.0 mL min−1. A series of eleven near-monodisperse poly(methyl methacrylate) standards (Mp values ranging from 800 to 988[thin space (1/6-em)]000 g mol−1) were used for column calibration.

Dynamic light scattering (DLS)

DLS studies were performed using a Zetasizer Nano ZS instrument (Malvern Instruments, UK) at a fixed scattering angle of 173°. Copolymer dispersions were diluted in n-dodecane (0.10% w/w) prior to light scattering studies at 25 °C. The intensity-average diameter and polydispersity of the diblock copolymer nanoparticles were calculated by cumulants analysis of the experimental correlation function using Dispersion Technology Software version 6.20. Data were averaged over ten runs each of thirty seconds duration. It is emphasized that DLS assumes a spherical morphology and reports intensity-average diameters. Thus, the apparent DLS diameter determined for highly anisotropic particles such as worms is neither equal to the worm length nor the worm width. Despite this limitation, DLS can be used to monitor a thermally-induced worm-to-sphere transition by determining the reduction in the apparent diameter.23

Transmission electron microscopy (TEM)

TEM studies were conducted using a FEI Tecnai G2 spirit instrument operating at 80 kV and equipped with a Gatan 1k CCD camera. Diluted diblock copolymer dispersions (0.10% w/w) were placed on carbon-coated copper grids and exposed to ruthenium(VIII) oxide vapor for 7 min at 20 °C prior to analysis.59 This heavy metal compound acted as a positive stain for the core-forming block to improve contrast. The ruthenium(VIII) oxide was prepared as follows: ruthenium(IV) oxide (0.30 g) was added to water (50 g) to form a black slurry; addition of sodium periodate (2.0 g) with continuous stirring produced a yellow solution of ruthenium(VIII) oxide within 1 min. In order to study the thermally-induced worm-to-vesicle transition, a sample vial containing 1.0 g of a 25% w/w PSMA9-PHPMA70 dispersion in mineral oil was placed in a pre-heated oil bath at 150 °C, allowed to equilibrate for 1 h, diluted with n-dodecane (preheated to the same temperature), and then a single droplet of this hot dispersion was transferred onto a TEM grid and allowed to evaporate.

Small-angle X-ray scattering (SAXS)

SAXS patterns were collected at a synchrotron source (Diamond Light Source, station I22, Didcot, UK) using monochromatic X-ray radiation (wavelength λ = 0.100 nm, with q ranging from 0.017 to 2.1 nm−1, where q = 4π[thin space (1/6-em)]sin[thin space (1/6-em)]θ/λ is the length of the scattering vector and θ is one-half of the scattering angle) and a 2D Pilatus 2 M pixel detector (Dectris, Switzerland). A glass capillary of 2.0 mm diameter was used as a sample holder. Scattering data were reduced using standard routines from the beamline and were further analyzed using Irena SAS macros for Igor Pro.60

Oscillatory rheology

An Anton Paar MCR 502 rheometer (equipped with TruGap functionality for online monitoring of the geometry gap), a variable-temperature Peltier plate, Peltier hood and a 50 mm 2° stainless cone was used for the rheology experiments. The storage (G′) and loss (G′′) moduli were determined as a function of temperature at a heating rate of 2 °C min−1, a fixed strain amplitude of 1.0%, and an angular frequency of 10 rad s−1. The sample gap was 207 μm.

Optical microscopy (OM)

Optical microscopy images were recorded using a Cole–Palmer compound optical microscope equipped with an LCD tablet display and a Moticam BTW digital camera.

Fluorescence microscopy

Fluorescence microscopy images of the w/o precursor emulsion and various w/o/w double Pickering emulsions were recorded using a Zeiss Axio Scope A1 microscope equipped with an AxioCam 1Cm1 monochrome camera. Nile Red dye was dissolved in the mineral oil prior to high-shear homogenization and the resulting oil droplets were imaged using Zeiss filter set 43 HE (excitation 550/25 nm and emission 605/70 nm). Images were captured and processed using ZEN lite 2012 software.

Laser diffraction

Each w/o/w emulsion was sized by laser diffraction using a Malvern Mastersizer 3000 instrument equipped with a hydro EV wet sample dispersion unit, a red HeNe laser operating at 633 nm and a LED blue light source operating at 470 nm. The stirring rate was adjusted to 1500 rpm in order to avoid creaming of the emulsion droplets during analysis. After each measurement, the cell was rinsed once with ethanol and three times with deionized water and the laser was aligned centrally to the detector prior to data acquisition.

Results and discussion

Synthesis of PSMA9 macro-CTA

The RAFT solution polymerization of SMA was conducted in toluene at 70 °C using CPDB as a RAFT CTA to produce the desired PSMA macro-CTA precursor (see Scheme 1). A preliminary kinetic study when targeting a PSMA5 macro-CTA had indicated first-order kinetics after an initial induction period of approximately 1 h and the expected linear evolution in molecular weight with conversion (see Fig. S1). In order to avoid the possible loss of RAFT chain-ends under monomer-starved conditions, the scaled-up SMA polymerization was quenched after 4 h by exposure to air, which resulted in an SMA conversion of 77%. This protocol produced approximately 26 g of PSMA homopolymer with a mean degree of polymerization (DP) of 9 and a relatively narrow molecular weight distribution (Mw/Mn = 1.12), indicating that good RAFT control was achieved.
image file: d0py00562b-s1.tif
Scheme 1 Synthesis of a poly(stearyl methacrylate) (PSMA) precursor via RAFT solution polymerization in toluene using 2-cyano-2-propyl benzodithioate (CPDB) at 70 °C, followed by the RAFT dispersion polymerization of 2-hydroxypropyl methacrylate (HPMA) in mineral oil at 90 °C.

Kinetic study of the RAFT dispersion polymerization of HPMA in mineral oil

In situ 1H NMR spectroscopy experiments were performed to examine the kinetics of the RAFT dispersion polymerization of HPMA at 90 °C when targeting PSMA9-PHPMA150 vesicles at 15% w/w solids. HPMA conversions were determined by comparing the integrated monomer vinyl proton signals at 5.0–6.0 ppm to the three methyl protons assigned to the toluene external standard at 2.3 ppm (see Fig. 1a). The HPMA polymerization proceeded relatively slowly for the first 10 min prior to an approximate six-fold rate enhancement. This time point corresponded to 29% HPMA conversion or a PHPMA DP of 44 and is attributed to the onset of micellar nucleation as the growing PHPMA chains become insoluble in the HPMA/mineral oil reaction mixture.28,32 Thereafter, first-order kinetics were observed up to 84% HPMA conversion, followed by a slower rate of polymerization under monomer-starved conditions (see Fig. 1b). More than 98% HPMA conversion was achieved within 70 min at 90 °C. This indicates a significantly faster polymerization than most previously studied PISA formulations in non-polar media, which is attributed to the polar nature of HPMA.24 In order to confirm this hypothesis, we compared the kinetics of this RAFT dispersion polymerization with that for a non-polar monomer, benzyl methacrylate (BzMA), while targeting PSMA9-PBzMA150 vesicles in mineral oil under precisely the same reaction conditions (i.e. using identical monomer and PSMA9 concentrations). For PSMA9-PHPMA150 vesicles, an HPMA conversion of 94% was achieved within 40 min whereas only 37% BzMA conversion was achieved for PSMA9-PBzMA150 vesicles on the same timescale (see Fig. 2). Moreover, the corresponding semilogarithmic plots suggested a pseudo-first order rate constant for the HPMA polymerization that was twelve-fold greater than that for the BzMA polymerization (see Fig. S2).
image file: d0py00562b-f1.tif
Fig. 1 (a) Selected partial 1H NMR spectra recorded during the RAFT dispersion polymerization of HPMA targeting a 15% w/w dispersion of PSMA9-PHPMA150 vesicles in mineral oil at 90 °C: after 3 min (black data), 12 min (red data), 15 min (blue data) and 19 min (green data) using toluene in d6-DMSO as an external standard. (b) Conversion vs. time curve (blue circles) and corresponding semi-logarithmic plot (red triangles) for the same PISA synthesis.

image file: d0py00562b-f2.tif
Fig. 2 Conversion vs. time curves obtained for the RAFT dispersion polymerization of either HPMA (blue circles) or BzMA (green squares) at 90 °C in mineral oil targeting either PSMA9-PHPMA150 vesicles at 15% w/w solids or PSMA9-PBzMA150 at 18% w/w solids, respectively. Conditions: [HPMA]0 = [BzMA]0 = 0.78 M; [PSMA9] = 17 g dm−3; [PSMA9]/[T21s] molar ratio = 5.0.

THF GPC analysis of the final PSMA9-PHPMA150 (98% conversion after 70 min; Mn = 19[thin space (1/6-em)]500 g mol−1, Mw/Mn = 1.24) and PSMA9-PBzMA150 diblock copolymers (97% conversion after 150 min; Mn = 17[thin space (1/6-em)]300 g mol−1, Mw/Mn = 1.11) indicated good RAFT control over each polymerization. Thus it appears that the RAFT dispersion polymerization of HPMA offers an advantage over the equivalent synthesis using BzMA.

Synthesis of PSMA9-PHPMAx diblock copolymer nanoparticles

A series of PSMA9-PHPMAx diblock copolymer nano-objects were then targeted via RAFT dispersion polymerization of HPMA in mineral oil at 90 °C. The PSMA9 precursor was utilized to polymerize HPMA and PHPMA DPs from 30 to 150 were targeted while varying the overall solids content between 15% and 30% w/w. In each case, more than 98% HPMA monomer conversion was achieved within 5 h as judged by 1H NMR spectroscopy. GPC analysis indicated that relatively good RAFT control was achieved in all cases (Mw/Mn ≤ 1.37, see Fig. 3). Targeting higher PHPMA DPs resulted in a systematic shift in the GPC curves towards higher molecular weight (see Fig. 3a), while minimal PSMA9 precursor contamination indicated high blocking efficiencies. Moreover, a linear evolution of Mn with target PHPMA DP is observed in Fig. 3b for a series of PSMA9-PHPMAx nano-objects prepared at 15% w/w solids, although a gradual increase in Mw/Mn is discernible as higher PHPMA DPs are targeted.
image file: d0py00562b-f3.tif
Fig. 3 (a) THF gel permeation chromatograms (vs. poly(methyl methacrylate) standards) obtained for the precursor PSMA9 macro-CTA (black dashed curve) and four PSMA9-PHPMAx diblock copolymers prepared via RAFT dispersion polymerization of HPMA in mineral oil at 90 °C targeting 15% w/w solids. (b) Plots of Mn (red squares) and Mw/Mn (blue circles) vs. PHPMA DP for a series of PSMA9-PHPMAx diblock copolymer nano-objects. The black dashed line indicates the theoretical Mn vs. PHPMA DP relationship for this series, with the difference being attributed to the GPC calibration error incurred by using poly(methyl methacrylate) standards.

Recently, we reported the chain extension of PSMA13 and PSMA18 stabilizer blocks using glycidyl methacrylate in mineral oil, but only kinetically-trapped spherical nanoparticles could be obtained.29 In contrast, using the shorter PSMA9 stabilizer block for the RAFT dispersion polymerization of HPMA provides convenient access to worms and vesicles, as well as spheres. Accordingly, a pseudo-phase diagram was constructed to facilitate the reproducible synthesis of such nano-objects (see Tables S1–S4), with copolymer morphology assignments being made on the basis of TEM studies (see Fig. 4).12,22,28,37 Well-defined spherical nanoparticles could be obtained at all copolymer concentrations examined, with DLS studies indicating narrow size distributions (polydispersity index, PDI ≤ 0.20) and a systematic increase in the intensity-average diameter when targeting higher PHPMA DPs, as expected.28,29 However, a pure worm phase could only be obtained at relatively high copolymer concentrations (either 25% w/w or 30% w/w solids). Derry et al. reported similar observations for poly(lauryl methacrylate)-poly(benzyl methacrylate) diblock copolymer nano-objects prepared in mineral oil.12 The rather broad mixed phase observed in Fig. 4e is similar to that recently reported by Rymaruk et al.31 for polydimethylsiloxane-poly(2-(dimethylamino)ethyl methacrylate) nano-objects in silicone oil. This suggests that sphere-sphere fusion is not particularly efficient for such PISA formulations. Nevertheless, pure vesicles could be accessed by targeting PHPMA DPs of 115 or higher at copolymer concentrations ranging from 15% w/w to 25% w/w. Interestingly, a mixed phase comprising vesicles and lamellae was observed when increasing the copolymer concentration up to 30% w/w (see Fig. 4d).


image file: d0py00562b-f4.tif
Fig. 4 Representative TEM images obtained for (a) PSMA9-PHPMA50 spheres, (b) PSMA9-PHPMA70 worms, (c) PSMA9-PHPMA120 vesicles and (d) a mixed phase comprising PSMA9-PHPMA120 lamellae and vesicles. (e) Pseudo-phase diagram constructed for PSMA9-PHPMAx diblock copolymer nano-objects prepared by RAFT dispersion polymerization of HPMA in mineral oil using a PSMA9 precursor and T21s initiator at 90 °C ([PSMA9]/[T21s] molar ratio = 5.0). [Black triangles (▲), red diamonds (image file: d0py00562b-u1.tif) and blue circles (image file: d0py00562b-u2.tif) represent pure vesicles, worms and spheres, respectively. Green squares (image file: d0py00562b-u3.tif) correspond to either sphere/worm or worm/vesicle mixed phases, whereas purple squares (image file: d0py00562b-u4.tif) represent a vesicle/lamellae mixed phase].

To confirm the copolymer morphologies assigned by TEM analysis, SAXS patterns were recorded for 1.0% w/w dispersions of four PSMA9-PHPMAx diblock copolymers originally prepared at 25% w/w (see Fig. 5). For PSMA9-PHPMA50, an approximate zero gradient was observed at low q as expected for spherical nanoparticles, and the local minimum in the scattering pattern at q ∼0.5 nm−1 indicates a mean core radius of approximately 9 nm.61 Fitting this SAXS pattern to a well-known spherical micelle model62 indicated an overall sphere diameter (Dsphere) of 21.1 ± 1.9 nm and a mean aggregation number (Nagg, or number of copolymer chains per nanoparticle) of 200 (see Table 1). This volume-average Dsphere value is consistent with the intensity-average diameter of 31 nm reported by DLS for these PSMA9-PHPMA50 spheres. The SAXS pattern recorded for PSMA9-PHPMA70 exhibits a gradient of approximately −1 in the low q region, which is consistent with the worm morphology indicated by TEM analysis. In this case, the local minimum observed at q ∼0.4 nm−1 represents the mean worm core radius. Fitting this SAXS pattern to a worm-like micelle model62 indicates an overall worm thickness (Tworm) of 22.5 ± 2.4 nm, a mean worm length (Lworm) of ∼252 nm and an Nagg of 3800. Comparing this aggregation number with that determined for the PSMA9-PHPMA50 spheres suggests that, on average, each worm is formed by stochastic 1D fusion of no more than 19 spheres. SAXS patterns recorded for PSMA9-PHPMA120 and PSMA9-PHPMA150 both exhibit low q gradients of approximately −2, which is consistent with a vesicular morphology.28,37 In this case, the subtle feature observed at low q (q ∼0.02 nm−1) indicates the overall vesicle radius while the well-defined local minimum at high q (q ∼0.2–0.3 nm−1) provides information regarding the vesicle membrane thickness. Fitting these patterns to a well-known vesicle model63 indicated that PSMA9-PHPMA120 and PSMA9-PHPMA150 vesicles exhibit comparable overall diameters (Dvesicle = 348 ± 54 nm and 366 ± 58 nm, respectively), but increasing the PHPMA DP from 120 to 150 led to a thicker vesicle membrane (Tmembrane = 16.0 ± 1.6 nm vs. 18.1 ± 1.9 nm, respectively). These data are consistent with the vesicle growth mechanism proposed by Warren et al.64 and later validated by Derry et al.28 for PISA syntheses. Mean Nagg values for PSMA9-PHPMA120 and PSMA9-PHPMA150 vesicles were calculated to be 177[thin space (1/6-em)]400 and 177[thin space (1/6-em)]500, respectively. These Nagg values are remarkably similar, which suggests that little or no net copolymer chain exchange occurs during the latter stages of the PISA synthesis of PSMA9-PHPMA150 vesicles (i.e. from 80% to 100% HPMA conversion).


image file: d0py00562b-f5.tif
Fig. 5 Small-angle X-ray scattering patterns recorded for 1.0% w/w dispersions of PSMA9-PHPMAx diblock copolymer spheres, worms and vesicles in mineral oil. Dotted lines indicate the data fits obtained using the relevant scattering model. In each case, these diblock copolymer nano-objects were initially synthesized at 25% w/w solids.
Table 1 Summary of nano-object dimensions obtained from fitting small-angle X-ray scattering patterns using either a spherical micelle,62 worm-like micelle62 or vesicle model.63 Dsphere is the overall sphere diameter such that Dsphere = 2Rs + 4Rg, where Rs is the mean core radius and Rg is the radius of gyration of the stabilizer chains. Tworm is the overall worm thickness (Tworm = 2Rwc + 4Rg, where Rwc is the mean worm core radius) and Lworm is the mean worm contour length. Dvesicle is the overall vesicle diameter (Dvesicle = 2Rm + Tmembrane + 4Rg, where Rm is the distance from the centre of the vesicle to the centre of the vesicle membrane, and Tmembrane is the vesicle membrane thickness). Nagg is the mean aggregation number (i.e. the mean number of copolymer chains per nano-object)
Block copolymer Nanoparticle morphology Dsphere Tworm Lworm Dvesicle Tmembrane Nagg
PSMA9-PHPMA50 Spheres 21.1 ± 1.9 nm 200
PSMA9-PHPMA70 Worms 22.5 ± 2.4 nm 252 nm 3800
PSMA9-PHPMA120 Vesicles 348 ± 54 nm 16.0 ± 1.6 nm 177[thin space (1/6-em)]400
PSMA9-PHPMA150 Vesicles 366 ± 58 nm 18.1 ± 1.9 nm 177[thin space (1/6-em)]500


Rheological studies of a PSMA9-PHPMA70 worm gel

The PSMA9-PHPMA70 worms prepared at 25% w/w solids in mineral oil form a free-standing gel owing to multiple inter-worm contacts.45 This worm gel was characterized by variable temperature oscillatory rheology, as described previously for other worm gel systems.23,35,40 Degelation occurred on heating above approximately 100 °C, with this critical temperature corresponding to the cross-over point for the bulk modulus G′ and storage modulus G′′ curves (see Fig. 6a). Similar thermoresponsive behavior has been reported for poly(lauryl methacrylate)-poly(benzyl methacrylate) nano-objects in n-dodecane and explained in terms of a worm-to-sphere transition owing to surface plasticization of the worm cores by ingress of hot solvent.23 However, in the present case TEM studies suggest that a partial worm-to-vesicle transition occurs on heating. The initial pure worms – diluted at 20 °C for TEM analysis (see Fig. 6b) – were transformed into a mixed phase comprising large vesicles and some remaining worms on heating up to 150 °C (see Fig. 6c). These observations are consistent with DLS studies: the sphere-equivalent intensity-average diameter determined at 20 °C for the initial dilute dispersion of pure worms (156 nm, PDI = 0.54) increased significantly after heating up to 150 °C (231 nm, PDI = 0.63). On cooling, regelation occurred at a critical gelation temperature (CGT) of approximately 100 °C, which suggests thermoreversible behavior. However, the initial G′ value of ∼ 6700 Pa was reduced by more than an order of magnitude to ∼420 Pa after this single thermal cycle. Moreover, TEM analysis of the diluted copolymer dispersion revealed a mixed morphology of worms and vesicles at 20 °C, thus indicating that the original pure worm morphology had not been restored on cooling (see Fig. S3).
image file: d0py00562b-f6.tif
Fig. 6 (a) Temperature dependence for the storage moduli (G′, red squares) and loss moduli (G′′, blue circles) observed for a PSMA9-PHPMA70 worm gel prepared at 25% w/w solids in mineral oil on heating from 20 to 180 °C (filled symbols) and cooling from 180 to 20 °C (hollow symbols) at a rate of 2.0 °C min−1. Data were recorded at 1.0% strain amplitude using an angular frequency of 10 rad s−1. Representative TEM images obtained for this PSMA9-PHPMA70 dispersion after dilution (b) at 20 °C (pure worms) and (c) 150 °C (mixed phase comprising vesicles and worms). This partial morphological transition accounts for the degelation that is observed above 100 °C.

Unlike previous morphological transformations of diblock copolymer nano-objects in non-polar media,23,27,65,66 this partial worm-to-vesicle transition on heating cannot be explained by surface plasticization of the PHPMA worm cores by hot solvent. This can only result in an increase in the volume of the solvophilic block relative to the solvophobic block, resulting in a reduction in the critical packing parameter (P) and thus favoring the formation of spheres. Instead, an increase in P is required to generate vesicles from the initial worms, which means that the relative volume of the solvophobic PHPMA block must increase relative to that of the solvophilic PSMA block. In principle, this could occur via uniform solvation of the core-forming PHPMA block. However, this seems unlikely given that even hot n-dodecane is likely to remain a very poor solvent for the hydroxyl-functional PHPMA chains. An alternative explanation could be that the poly(lauryl methacrylate) block becomes less solvated at elevated temperature and hence occupies a smaller volume relative to that at 20 °C. Clearly, further studies are warranted to provide a satisfactory physical explanation for this unexpected morphological transition, which serves to demonstrate that there is still much to learn about such thermoresponsive PISA formulations.

PSMA9-PHPMA50 diblock copolymer spheres as putative Pickering emulsifiers

PSMA9-PHPMA50 diblock copolymer spheres prepared at 15% w/w copolymer concentration in mineral oil were evaluated as a putative Pickering emulsifier. In some respects such nanoparticles are quite similar to the PSMA14-PNMEP49 diblock copolymer spheres prepared in n-dodecane by Cunningham et al.24 In both cases the PSMA stabilizer block is relatively short and the core-forming block is relatively polar, with PNMEP being water-soluble67 and PHPMA being only weakly hydrophobic.68

Cunningham et al.24 reported that hydrophobic PSMA14-PNMEP49 spherical nanoparticles unexpectedly formed oil-in-water Pickering emulsions when subjected to high-shear homogenization with water. This observation was attributed to in situ inversion of the diblock copolymer nanoparticles to produce hydrophilic PNMEP49-PSMA14 spheres.24 In contrast, when emulsification was conducted under low shear (via hand-shaking), the anticipated water-in-oil Pickering emulsion was formed. Furthermore, using relatively low water volume fractions (e.g. ≤0.25) also enabled the preparation of water-in-oil emulsions. There are two key differences between the current study and that reported by Cunningham et al.24 Firstly, the current diblock copolymer spheres are prepared in mineral oil rather than n-dodecane. Perhaps more importantly, the nanoparticle cores are composed of PHPMA chains, which – unlike PNMEP – do not become hydrophilic under any conditions. Hence inversion of the diblock copolymer chains to form PHPMA50-PSMA9 nanoparticles is not expected to occur in the present study, which should lead to differing Pickering emulsifier behavior.

First, the effect of varying the water volume fraction on Pickering emulsion formation was studied. A series of emulsions was prepared via high-shear homogenization at 13[thin space (1/6-em)]500 rpm for 2 min using a fixed 1.0% w/w dispersion of PSMA9-PHPMA50 spheres in mineral oil and water volume fractions ranging from 0.135 to 0.875 (see Scheme 2). Fig. S4 shows digital photographs of the physical appearance of the resulting emulsions, which were visualized using optical and fluorescent microscopy (see Fig. 7). The digital photograph of the emulsion prepared at a water volume fraction of 0.25 indicates the formation of a water-in-oil emulsion because the denser water droplets sediment to form the lower phase, which has a smaller volume. Optical microscopy images recorded for this emulsion indicated a mean droplet diameter of around 25 μm. To aid identification of the emulsion type, Nile Red dye was dissolved in the mineral oil prior to homogenization to enable fluorescence microscopy studies. As shown in Fig. 7d, this fluorescent label is mainly located in the continuous phase, confirming that a water-in-oil emulsion is obtained in this case. In contrast, an emulsion prepared at a water volume fraction of 0.50 comprised relatively large fluorescently-labelled droplets, suggesting formation of an oil-in-water emulsion under such conditions, see Fig. 7e. However, closer inspection revealed that spherical water droplets were present within these coarse oil droplets, indicating that a water-in-oil-in-water (w/o/w) double emulsion had been formed. Literature examples of the one-step formation of such complex emulsions using a single emulsifier are known but are relatively rare.69–74 Employing a water volume fraction of 0.75 also produced a w/o/w double emulsion, albeit with significantly fewer encapsulated aqueous droplets, as shown in Fig. 7f. Increasing the water volume fraction to 0.875 appeared to produce a simple oil-in-water emulsion, as judged by optical microscopy (see Scheme 2). However, fluorescence microscopy studies indicated the presence of encapsulated aqueous droplets within at least some of the oil droplets, indicating that a w/o/w double emulsion was in fact obtained (see Fig. S5). Presumably, simple o/w emulsions cannot be formed under such conditions because, unlike PNMEP, the PHPMA block is not hydrophilic so nanoparticle inversion cannot occur.24


image file: d0py00562b-s2.tif
Scheme 2 Schematic representation of the three types of Pickering emulsions that could be formed when using PSMA9-PHPMA50 spherical nanoparticles synthesized directly in mineral oil via RAFT dispersion polymerization of HPMA. In principle, systematic variation of the volume fraction of the aqueous phase might enable the preparation of water-in-oil, water-in-oil-in-water or oil-in-water emulsions. In practice, the latter type of emulsion cannot be formed by these nanoparticles (see main text).

image file: d0py00562b-f7.tif
Fig. 7 (a–c) Optical microscopy images and (d–f) fluorescence microscopy images recorded for Pickering emulsions prepared by high-shear homogenization of 1.00% w/w PSMA9-PHPMA50 spheres in mineral oil at 13[thin space (1/6-em)]500 rpm using water volume fractions (Vw) of 0.25, 0.50 or 0.75. In each case, the oil phase has been labelled with a water-insoluble dye (Nile Red). For a water volume fraction of 0.25, black aqueous droplets are observed within a continuous phase comprising the mineral oil and this red dye. However, when emulsions are prepared at a water volume fraction of 0.50, red oil droplets are observed within a dark aqueous continuous phase. Moreover, water droplets are visible within these dyed oil droplets, indicating the formation of w/o/w double emulsions. Similar w/o/w double emulsions are produced at a water volume fraction of 0.75, albeit containing fewer encapsulated aqueous droplets.

According to the literature, high-shear homogenization of diblock copolymer nano-objects can cause in situ dissociation in some cases, which leads to the formation of emulsion droplets stabilized by individual diblock copolymer chains acting as a polymeric surfactant.52,53,55,75 Moreover, such instances typically involve PHPMA-core nanoparticles, albeit those prepared via aqueous PISA formulations rather than the present non-aqueous formulation.52,53,55 Fortunately, whether such nano-object dissociation actually occurs in practice can be assessed by determining the effect of varying the copolymer concentration on the mean droplet diameter.53,55,70,76 To examine whether genuine Pickering emulsions are formed in the present study, the copolymer concentration was systematically varied between 0.03% and 1.00% w/w in mineral oil. These dispersions were homogenized using a water volume fraction of 0.75, 0.50 or 0.25 at a constant shear rate of 13[thin space (1/6-em)]500 rpm to produce a series of either w/o single emulsions or w/o/w double emulsions. Fig. S6 shows a representative digital photograph recorded for the resulting emulsions. Droplet size distributions for the w/o/w emulsions were analyzed using laser diffraction, as shown in Fig. 8. Systematically increasing the copolymer concentration led to a gradual reduction in the mean droplet diameter until a limiting value of approximately 25 μm was attained at 1.00% w/w [PSMA9-PHPMA50]. Similar results were obtained for water-in-oil emulsions prepared at a water volume fraction of 0.25 (see Fig. S7). These observations are consistent with the corresponding optical microscopy images (see inset) and indicate that the nanoparticles remain intact after high-shear homogenization, leading to the formation of genuine Pickering emulsions.52,53,55,75


image file: d0py00562b-f8.tif
Fig. 8 Variation in volume-average droplet diameter (determined by laser diffraction studies) with copolymer concentration obtained for water-in-oil-in-water (w/o/w) Pickering double emulsions produced via high-shear homogenization (13[thin space (1/6-em)]500 rpm for 2 min at 20 °C) of PSMA9-PHPMA50 diblock copolymer spheres in mineral oil at a constant water volume fraction of 0.75. Inset: representative optical microscopy images recorded for such w/o/w emulsions prepared using copolymer concentrations of 0.0625, 0.25 or 1.00% w/w, respectively.

To examine the effect of copolymer concentration on the formation of Pickering double emulsions, a series of emulsions were prepared at a constant water volume fraction of 0.50 using a shear rate of 13[thin space (1/6-em)]500 rpm for 2 min. The resulting emulsion droplets were imaged using fluorescence microscopy, see Fig. 9. Preparing the emulsions at 0.03% w/w copolymer concentration produced relatively large w/o single emulsions of approximately 150 μm diameter, as confirmed by the presence of the Nile Red label in the continuous phase. Increasing the copolymer concentration up to 0.125% w/w led to a systematic reduction in the mean droplet diameter to around 20 μm (see Fig. 9b), suggesting that the nanoparticles also survive high-shear homogenization when prepared at a water volume fraction of 0.50. However, increasing the copolymer concentration further led to the formation of w/o/w double emulsions, see Fig. 9c. Thus, either w/o single emulsions or w/o/w double emulsions can be prepared at a constant water volume fraction by simply varying the copolymer concentration.


image file: d0py00562b-f9.tif
Fig. 9 Fluorescence microscopy images recorded for emulsions prepared using PSMA9-PHPMA50 spheres at a copolymer concentration of (a) 0.03% w/w, (b) 0.125% w/w or (c) 0.50% w/w. In each case, the water volume fraction was fixed at 0.50 and a constant stirring rate of 13[thin space (1/6-em)]500 rpm was used for 2 min during high-shear homogenization at 20 °C.

Finally, the effect of varying the shear rate on emulsion formation was examined. A series of four emulsions were prepared at a constant copolymer concentration of 1.00% w/w and a water volume fraction of 0.50 using shear rates ranging from 3500 rpm to 20[thin space (1/6-em)]000 rpm. Nile Red was again used to label the mineral oil prior to emulsification to facilitate fluorescence microscopy studies. For emulsions prepared at relatively low shear (i.e., 3500 or 7500 rpm), this dye is located within the continuous phase, confirming the formation of w/o emulsions, see Fig. S8. As anticipated, w/o/w emulsions are formed at 15[thin space (1/6-em)]500 rpm, with dark aqueous domains being clearly visible within the dyed oil droplets. Finally, a shear rate of 20[thin space (1/6-em)]000 rpm did not produce significantly smaller oil droplets but there is some evidence for a higher density of encapsulated water domains within the oil droplets.

Conclusions

The PISA synthesis of PSMA-PHPMA diblock copolymer spheres, worms and vesicles was achieved via RAFT dispersion polymerization of HPMA at 90 °C in mineral oil. In situ 1H NMR spectroscopy was utilized to examine the kinetics of the PISA synthesis of PSMA9-PHPMA150 vesicles, for which (more than 98%) monomer conversion was achieved within 70 min. This is a remarkably short time scale compared to most other PISA formulations conducted in non-polar media and is attributed to the relatively polar nature of the HPMA monomer. Construction of a pseudo-phase diagram enables the reproducible targeting of PSMA9-PHPMAn spheres, worms or vesicles, as confirmed by TEM, DLS and SAXS studies. Thus this is a rare example of the use of a commercially available polar monomer for PISA syntheses in non-polar media that offers access to the full range of copolymer morphologies. The worms formed thermoresponsive free-standing gels with degelation occurring on heating above 100 °C. Unusually, such degelation is the result of a worm-to-vesicle transition, rather than a worm-to-sphere transition. Finally, PSMA9-PHPMA50 spheres were evaluated as Pickering emulsifiers. Water-in-oil emulsions were obtained at relatively low water volume fractions (0.125 to 0.375). At higher volume fractions (0.50 to 0.875), a series of water-in-oil-in-water Pickering double emulsions were obtained with progressively fewer encapsulated aqueous droplets. Using either higher shear rates or a higher copolymer concentration during homogenization also favoured the formation of such double emulsions.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We thank EPSRC for CDT PhD studentships to support C. G. and S. J. H. (EP/L016281) and The Lubrizol Corporation Ltd for further financial support of this project and for permission to publish these results. S. P. A. acknowledges an EPSRC Established Career Particle Technology Fellowship (EP/R003009) and also the Leverhulme Trust (RPG-2016-330) for post-doctoral support of M. J. D. Dr S. van Meurs is thanked for her assistance with the in situ NMR experiments. The authors thank C. J. Hill and Dr S. Tzokov at the University of Sheffield Biomedical Science Electron Microscopy suite. We gratefully acknowledge Diamond Light Source for granting synchrotron beamtime (SM14892) and thank the personnel of I22 for their kind assistance.

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Footnotes

Electronic supplementary information (ESI) available: PSMA polymerization kinetics; semi-logarithmic plots for RAFT dispersion polymerization of HPMA and BzMA; summary tables of copolymer characterization; further TEM evidence for the partial worm-to-vesicle transition of PSMA9-PHPMA70 worms obtained after oscillatory rheology studies; digital photographs of the visual appearance of the Pickering emulsions; further fluorescence microscopy evidence for a w/o/w Pickering double emulsion; droplet diameter vs. copolymer concentration plot for w/o Pickering emulsions. See DOI: 10.1039/d0py00562b
Present address: Aston Institute of Materials Research, Aston University, Birmingham, B4 7ET, UK.

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