How Does Cross-Linking Affect the Stability of Block Copolymer


Dec 15, 2011 - Block copolymer vesicles are conveniently prepared directly in water at relatively high solids by polymerization-induced self-assembly ...
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How Does Cross-Linking Affect the Stability of Block Copolymer Vesicles in the Presence of Surfactant? P. Chambon,† A. Blanazs,† G. Battaglia,‡ and S. P. Armes†,* †

Dainton Building, Department of Chemistry, The University of Sheffield, Brook Hill, Sheffield, South Yorkshire, S3 7HF United Kingdom ‡ Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN United Kingdom S Supporting Information *

ABSTRACT: Block copolymer vesicles are conveniently prepared directly in water at relatively high solids by polymerizationinduced self-assembly using an aqueous dispersion polymerization formulation based on 2-hydroxypropyl methacrylate. However, dynamic light scattering studies clearly demonstrate that addition of small molecule surfactants to such linear copolymer vesicles disrupts the vesicular membrane. This causes rapid vesicle dissolution in the case of ionic surfactants, with nonionic surfactants proving somewhat less destructive. To address this problem, glycidyl methacrylate can be copolymerized with 2-hydroxypropyl methacrylate and the resulting epoxy-functional block copolymer vesicles are readily cross-linked in aqueous solution using cheap commercially available polymeric diamines. Such epoxy-amine chemistry confers exceptional surfactant tolerance on the cross-linked vesicles and also leads to a distinctive change in their morphology, as judged by transmission electron microscopy. Moreover, pendent unreacted amine groups confer cationic character on these cross-linked vesicles and offer further opportunities for functionalization.



INTRODUCTION It is well documented that amphiphilic AB diblock copolymers can undergo self-assembly in aqueous solution to form various types of nanostructures.1−4 Simply tuning the hydrophilic/ hydrophobic balance (and hence relative chain volume fractions) of the diblock copolymer dictates the particle morphology. More specifically, gradually increasing the proportion of the hydrophobic block leads to a progressive evolution from spherical micelles5,6 to worm-like micelles2,7−9 to diblock copolymer vesicles (aka polymersomes).3,10−14 Block copolymer micelles are generally considered to be more robust than classical surfactant micelles,15−17 since the former exhibit much lower critical micelle concentrations18 and can often be regarded as kinetically frozen (or nonergodic) due to the relatively slow exchange of copolymer chains between micelles.19 Similarly, block copolymer vesicles offer significant advantages over their low molecular weight “liposome” analogues due to their thicker, tougher membranes12,20,21 and hence significantly reduced permeabilities.10,22 However, extensive processing (such as a pH23 or solvent switch1 or thin film rehydration19) is usually required to induce copolymer self-assembly, which normally results in the generation of vesicular nanoparticles at relatively high dilution (typically <1% solids).24 Despite these processing limitations, block copolymer vesicles have been intensively studied over the past decade, © 2011 American Chemical Society

particularly with regard to their potential as drug delivery vehicles.13,25−34 Moreover, the incorporation of stimulusresponsive character within the copolymer amphiphile can significantly enhance potential applications. For example, block copolymer vesicles have been designed to respond to a local oxidizing environment,35 temperature,36,37 or a reduction in pH.23,38−41 Diblock copolymers comprising pH-responsive poly[2-(diisopropylamino)ethyl methacrylate] (PDPA) and highly biocompatible poly[2-(methacryloyloxy)ethyl phosphorylcholine] (PMPC) have been evaluated for their potential use as nontoxic intracellular delivery vehicles.42,43 In recent in vivo mouse studies, these PMPC-PDPA vesicles were taken up by oral squamous carcinoma cells, suggesting a possible mechanism for the delivery of anticancer drugs.44 There are many literature examples of “two-component” block copolymer vesicles. Santore and co-workers45 reported the effect of adding a Pluronic-type block copolymer surfactant to giant vesicles prepared using a poly(ethylene oxide)poly(ethyl ethylene) (PEO-PEE) diblock copolymer. Weak, reversible penetration of the membrane by the copolymer surfactant increased the vesicle permeability by a factor of two and allowed the surfactant to act on both the inner and outer Received: November 17, 2011 Revised: December 14, 2011 Published: December 15, 2011 1196

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Figure 1. Schematic representation of the epoxy-amine cross-linking of PGMA55-(PHPMA247-stat-PGlyMA82) copolymer vesicles.

leaflets. Eisenberg’s group46 judiciously mixed two poly(acrylic acid)-polystyrene diblocks with approximately the same polystyrene block length but unequal poly(acrylic acid) block lengths to demonstrate the effect of polydispersity on membrane formation. The same team also reported the spatial segregation of polyacid and polybase blocks on the inner and outer leaflets using binary mixtures of poly(acrylic acid)polystyrene and poly(4-vinylpyridine)-polystyrene diblocks.47 Discher and co-workers48 have shown that mixtures of diblock copolymers with differing corona-forming blocks lead to nanophase separation to form Janus-type giant vesicles. Massignani et al.49 have extended this approach to include submicrometer-sized vesicles and demonstrated that the “patchy” nature of the vesicle surface profoundly influences their rate of cell uptake. Various mixtures of block copolymers and classical surfactants have also been explored. For example, addition of a surfactant to maintain block copolymer solubility can be used as a processing aid, with the weakly bound surfactant being displaced on dilution so as to induce vesicle formation.50 Pispas has briefly reviewed the use of ionic surfactants that bind electrostatically to the oppositely charged polyelectrolyte chains of a diblock copolymer to induce vesicle formation.51 Cross-linking within either the membrane core or the hydrophilic corona can significantly increase the stability of copolymer vesicles with respect to dilution, an external stimulus (e.g., a change in pH or temperature), a surfactant challenge or addition of a good solvent for both blocks. In principle, vesicle cross-linking can be achieved by ionic complexation. An example of this approach has been reported by Kataoka and co-workers,52 who used a binary mixture of a PEO-polyacid with a PEO-polybase to produce a vesicular polyion complex. Alternatively, cationic steric stabilizer chains can be cross-linked by addition of an anionic homopolyelectrolyte37 or gold nanoparticles.53 However, such ionically cross-linked vesicles are presumably rather sensitive to the presence of salt, due to screening of the electrostatic attractive forces. This problem can be circumvented by using covalent cross-linking to stabilize copolymer vesicles. The most common strategy involves cross-

linking within the vesicle membrane. If the hydrophobic block is polybutadiene, this can be achieved by photocross-linking.54−57 This approach has been modified by Katz and coworkers,58 who introduced a terminal vinyl group into a poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-PCL) diblock copolymer via acrylation of the hydroxyl end-group on the PCL chain. A membrane-soluble photoinitiator was subsequently employed to covalently stabilize the vesicles on exposure to uv light. Moreover, these cross-linked vesicles were highly resistant to a surfactant challenge using nonionic Triton X-100. Similarly, uv cross-linking of vesicles can be achieved via vinyl groups located at the terminus of the coronal stabilizer chains, rather than the membrane-forming chains.59 Intriguingly, Stenzel and co-workers60 have demonstrated that reversible addition−fragmentation chain transfer (RAFT) polymerization of a diacrylate monomer between the hydrophilic stabilizer block and the hydrophobic membrane-forming block can also be employed to produce covalently stabilized block copolymer vesicles. Chen and co-workers61 reported that poly(ethylene oxide)-block-poly[3-(trimethoxysilyl)propyl methacrylate] (PEO-PTMSPMA) copolymers initially form linear vesicles in aqueous solution, with in situ hydrolysis of the trimethoxysilyl groups leading to silicification of the membrane. Later, Du and Armes62 showed that statistical copolymerization of 2-(diethylamino)ethyl methacrylate (DEA) with the TMSPMA comonomer produced pH-responsive silicified PEO-P(DEA-stat-TMSPMA) copolymer vesicles. Of particular relevance to the present work, Zhu et al.63 exploited epoxydiamine chemistry to achieve covalent cross-linking within the membranes of poly(ethylene oxide)-block-poly(glycidyl methacrylate) diblock copolymer vesicles using alkyldiamines. Recently, Charleux and co-workers64−66 reported the efficient preparation of block copolymer vesicles directly in water via aqueous emulsion polymerization using living radical polymerization. We have recently extended this groundbreaking approach to include aqueous dispersion polymerization.67−69 The latter formulation has provided a unique insight into the mechanism of the worm-to-vesicle transition68 and has also allowed the construction of a detailed phase 1197

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Figure 2. (A) 1H NMR spectra of PGMA55-PHPMA330 and PGMA55-P(HPMA247-stat-GlyMA82) recorded in d6-DMSO. (B) GPC traces of PGMA55, PGMA55-PHPMA330, and PGMA55-P(HPMA247-stat-GlyMA82) (DMF eluent, 60 °C, flow rate = 1 mL min−1, PMMA calibration standards). In both cases, the samples were freeze-dried overnight prior to analysis in order to remove as much water as possible.

diagram,69which ensures that such vesicle syntheses are reproducible. Since block copolymer vesicles can now be conveniently and reproducibly prepared at 20−25% solids using industrially scalable chemistry, it is appropriate to consider their potential for the encapsulation and release of various actives. However, many commercial formulations in the home and personal care sector contain significant levels of small molecule surfactant(s), which may cause vesicle dissolution. Pata and coworkers70 studied the effect of addition of a single nonionic surfactant (Triton X-100) on the colloidal stability of vesicles prepared using three different poly(ethylene oxide)-based diblock copolymers, which enabled the membrane thickness to be systematically varied. For an arbitrary time period of ten minutes, the minimum surfactant concentration required for a

50% reduction in scattered light intensity as judged by dynamic light scattering increased linearly with membrane thickness and a model was proposed to account for these observations. As mentioned above, Katz and co-workers58 also examined the effect of addition of Triton X-100 on the colloidal stability of poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-PCL) vesicles. Light scattering investigations demonstrated that crosslinking confers an enhanced resistance to the surfactant and prevents the disruption of the vesicles. However, these two articles seem to be the only examples of the effect of added (non-ionic) surfactant on the colloidal stability of block copolymer vesicles in the literature. In the present work, we examine the extent to which block copolymer vesicles can tolerate the addition of either anionic, cationic or nonionic 1198

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surfactant, develop a robust covalent stabilization protocol based on cheap polymeric cross-linkers (see Figure 1) and also explore whether such cross-linking improves the tolerance of the vesicle membrane toward such surfactants.



EXPERIMENTAL SECTION

Materials. Glycerol monomethacrylate (GMA) was obtained from Cognis (Hythe, U.K.) and used as received. 2-Hydroxypropyl methacrylate (HPMA), glycidyl methacrylate (GlyMA), 4,4′-azobis(4-cyanopentanoic acid) (ACVA), 2-cyano-2-propylbenzodithioate, ethylenediamine, and bis(3-aminopropyl)-terminated poly(ethylene glycol) (PEG34 diamine, DP ≈ 34) were purchased from Aldrich and used as received. Jeffamines D-230, D-400, and D-2000 (230, 400, and 2000 g mol−1, respectively) were kindly donated by Huntsman Chemicals and used as received. Sodium dodecylbenzenesulfonate, didecyldimethylammonium chloride and hepta(ethylene glycol) monoalkyl ether (C12−C14) were used as received. Synthesis of Poly(glycerol monomethacrylate-block-2-hydroxypropyl methacrylate) (PGMA-PHPMA) Diblock Copolymer Vesicles. PGMA55 (8,800 g mol−1) macro-chain transfer agent (macro-CTA) was synthesized by RAFT polymerization using 2cyano-2-propyl dithiobenzoate as previously described.67,68 For the preparation of PGMA55-PHPMA330 diblock copolymer vesicles, PGMA55 macro-CTA (0.100 g, 0.01136 mmol) and 2-hydroxypropyl methacrylate (HPMA, 0.540 g, 3.75 mmol; target DP = 330) were degassed under nitrogen flow in a 25 mL round-bottomed flask equipped with a magnetic stirrer bar. After 15 min, the 4,4′-azobis(4cyanopentanoic acid) radical initiator (ACVA, 1.5 mg, 5.3 μmol, [macro-CTA]/[ACVA] = 2.0) was introduced into the flask, which was flushed with nitrogen for 5 min. Deionized water (6.4 mL, total solids content = 10 w/v %) (degassed under N2 prior to use) was then added and the mixture was further purged with nitrogen for less than 5 min. Finally, this reaction mixture was placed in an oil bath at 70 °C and stirred for 4 h. After cooling to room temperature, two aliquots were extracted for GPC and NMR analysis (see Figure 2 and Supporting Information). A PGMA55-P(HPMA247-stat-GlyMA82) diblock copolymer was also prepared via the same protocol using the following formulation: PGMA55 macro-CTA (0.200 g, 0.0227 mmol), 2-hydroxypropyl methacrylate (HPMA, 0.810 g, 5.625 mmol, 247 eq. based on macro-CTA), glycidyl methacrylate (GlyMA, 0.2665 g, 1.875 mmol, 82 eq. based on macro-CTA), 4,4′-azobis(4-cyanopentanoic acid) (ACVA, 3.0 mg, 5.3 μmol, [macro-CTA]/[ACVA] = 2.0), and deionized water (12.8 mL, 10.0 w/v % solids). Cross-Linking of PGMA-P(HPMA-stat-GlyMA) Diblock Copolymer Vesicles. To prepare cross-linked PGMA55-P(HPMA247stat-GlyMA82) diblock copolymer vesicles using one equivalent of Jeffamine D-230 per epoxy group (i.e., an amine/epoxy molar ratio of 2.0), PGMA55-P(HPMA247-stat-GlyMA82) vesicles at 10 w/v % solids were diluted ten-fold and 10.0 mL of this diluted aqueous dispersion was transferred to a sample tube containing 33.0 mg Jeffamine D-230 (i.e., [diamine]/[epoxy] molar ratio = 1.0, or [amine]/[epoxy] molar ratio = 2.0). The mixture was agitated using a vortex mixer for a few minutes then stirred overnight on a roller mixer at 20 °C. The resulting cross-linked vesicles were characterized by DLS, TEM and elemental microanalyses to determine their particle size and nitrogen content, respectively. In some cases, the cross-linked vesicles were purified in order to remove any unreacted Jeffamine cross-linker, either by dialysis against water (membrane molecular weight cutoff = 50 000 Da) or via three centrifugation/redispersion cycles. The resulting aqueous dispersion was freeze-dried overnight to obtain an off-white powder. This was analyzed by elemental microanalyses and the results were compared to those obtained for the unpurified dispersion. The above cross-linking protocol was also employed for the ethylene diamine, the higher molecular weight Jeffamine 400 and also the PEG 34 diamine (see Figure 3). Surfactant Challenge. In a typical surfactant challenge, 2.0 mL of a 1.0 w/v % aqueous dispersion of PGMA55-P(HPMA247-statGlyMA82) vesicles was added to 20 mg of sodium dodecylbenzenesul-

Figure 3. Chemical structures of the three surfactants and five diamine-based cross-linkers used in this study. fonate in a sample tube. This ad-mixture was then allowed to stir on a roller mixer room temperature for at least 16 h. The resulting copolymer/surfactant solution was subsequently characterized by DLS and TEM. The same protocol was used to examine both linear and cross-linked vesicles at various anionic surfactant concentrations. In further experiments, the effect of adding a cationic and a nonionic surfactant to such block copolymer vesicles was also explored (see Figure 3). Finally, the effect of addition of Jeffamine 230 and 400 to the linear PGMA-PHPMA diblock copolymer vesicles was explored. Copolymer Characterization. Gel Permeation Chromatography (GPC). Molecular weights and polydispersities were determined by GPC using the following set-up: Varian 290-LC pump injection module, a Varian 390-LC refractive index detector, two Polymer Laboratories PLgel 5 μm ‘Mixed C′ columns maintained at 60 °C and DMF eluent (containing 0.01 M LiBr) at a flow rate of 1.0 mL min−1. DMSO was employed as a flow rate marker. Calibration was achieved using a series of near-monodisperse poly(methyl methacrylate) standards. NMR Spectroscopy. 1H NMR spectra were recorded using a Bruker Avance 400 spectrometer operating at 400 MHz. Dynamic Light Scattering (DLS). DLS measurements were performed using a Malvern Zetasizer Nano ZS instrument. The intensity-average diameter, polydispersity and light scattering intensity were recorded for aqueous dispersions at a fixed copolymer concentration of 1.0 w/v %. Aqueous Electrophoresis. Zeta potentials were determined from mobilities using the same Malvern Zetasizer Nano ZS described above. The zeta potential, ζ, was calculated71 from the electrophoretic mobility (u) using the Smoluchowsky relationship, ζ = ηu/ε, where it is assumed that ka ≫ 1 (where η is the solution viscosity, ε is the dielectric constant of the medium, and k and a are the Debye−Hückel parameter and the particle radius, respectively). The background electrolyte was 2 × 10−3 M NaCl. Transmission Electron Microscopy (TEM). Diblock copolymer vesicles initially prepared at 10 w/v % solids were diluted fifty-fold at 20 °C to generate 0.20 w/v % aqueous dispersions. Copper/palladium TEM grids (Agar Scientific) were surface-coated in-house to produce a thin film of amorphous carbon. The grids were then plasma glowdischarged for 30 s to create a hydrophilic surface. Individual samples (0.20 w/v %, 12 μL) were adsorbed onto the fresh glow-discharged grids for one minute and then blotted with filter paper to remove excess solution. To stain the aggregates, uranyl formate (0.75 w/v %) solution (9 μL) was soaked on the sample-loaded grid for 20 s and then carefully blotted to remove excess stain. The grids were then dried using a vacuum hose. Imaging was performed on a Phillips CM100 instrument at 100 kV, equipped with a Gatan 1 k CCD camera. 1199

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Elemental Microanalyses. Carbon, hydrogen, and nitrogen contents were determined in-house using a Perkin-Elmer 2400 elemental analyzer.

at 10 w/v% solids, as described previously.68 After HPMA polymerization was allowed to continue for 4 h at 70 °C, almost full monomer conversion (>99%) was achieved as judged by 1H NMR spectroscopy. Both TEM and DLS studies of the resulting milky-white solution confirmed the formation of copolymer vesicles at 20 °C (see entry 1 in Table 1 and the Supporting Information). GPC measurements indicated the formation of a low-polydispersity block copolymer with relatively high blocking efficiency, which suggests minimal PGMA macro-CTA contamination (see Figure 2). Vesicle Challenge with Surfactant or Diamine. The colloidal stability of 1.0 w/v % aqueous solutions of these diblock copolymer vesicles was assessed with respect to either a surfactant or a diamine challenge (see Table 1 and Figure 3). The initial light scattering intensity of the vesicular solutions determined by DLS was compared to that obtained after addition of surfactant (or diamine). Thus a relatively low normalized intensity corresponds to a much more transparent (i.e., more weakly scattering) dispersion. The results presented in Table 1 confirm that a substantial loss in turbidity occurs on addition of relatively small amounts of either ionic surfactant or various diamines, which suggests vesicle destruction. Surprisingly, the addition of anionic surfactant led to a substantial increase in the apparent diameter (see Table 1). However, the final light scattering intensity is less than 1% of the original value, suggesting the formation of relatively loose ill-defined polymer−surfactant aggregates, rather than well-defined vesicles. This interpretation is consistent with the very high polydispersities observed in the presence of ionic surfactants. The anionic surfactant is clearly the most destructive additive, with essentially complete vesicle degradation being observed at concentrations as low as 0.50 w/v%. The cationic surfactant was not quite as effective at this concentration, but almost complete loss in scattering intensity was observed at 1.0 w/v%. However, addition of neutral surfactant produced only a moderate loss (∼20%) in scattering intensity even when employed at up to 10 w/v %. The colloidal aggregates present after a surfactant challenge using either 0.10 w/v % anionic surfactant, 0.50 w/v % cationic or 10.0 w/v % neutral surfactant were assessed by TEM (see the Supporting Information).



RESULTS AND DISCUSSION Various covalent cross-linking strategies have been reported for the preparation of diblock copolymer vesicles, including radical cross-linking of unsaturated groups,54−59 silicification,61,62 diacrylate cross-linking,60 amine/gold sol complexation,53 and epoxy-amine chemistry.63 In the present work, we utilized the latter approach (see Scheme 1) since this chemistry is Scheme 1. General Reaction between Amine and Epoxy Groups

compatible with our aqueous dispersion formulation. Moreover, both the glycidyl methacrylate comonomer and the Jeffamine polymeric diamines are cheap, commercially available reagents. This approach was used by Chen and co-workers72 to stabilize micelles, worms and bilayers and also by ourselves to produce latex-based colloidosomes.73 However, as far as we are aware, there has only been one previous report of the use of epoxy-amine chemistry for the stabilization of block copolymer vesicles.63 This protocol involved addition of diamine crosslinker to a poly(ethylene oxide)-block-poly(glycidyl methacrylate) molecularly dissolved in THF, prior to the addition of water to induce in situ self-assembly. Thus some epoxy-amine cross-linking is likely to occur prior to vesicle formation, which may account for the apparent vesicle aggregation indicated by TEM studies. In contrast, in the present work the diamine cross-linker is added after vesicle formation so as to minimize side-reactions. Synthesis of Linear PGMA-PHPMA Diblock Copolymer Vesicles. PGMA55-PHPMA330 diblock copolymer vesicles were synthesized via RAFT aqueous dispersion polymerization

Table 1. Effect of Addition of Either Surfactant or Diamine Cross-Linker on the Colloidal Stability of a 1.0 w/v % Aqueous Dispersion of Linear PGMA55-PHPMA330 Diblock Copolymer Vesicles entry 1 2

nature of surfactant or cross-linker additive none (i.e., pristine linear PGMA55-PHPMA330 vesicles) sodium dodecylbenzenesulfonate (anionic surfactant)

3

didecyldimethylammonium chloride (cationic surfactant)

4

alcohol ethoxylate (7EO C12−C14) (neutral surfactant) ethylenediamine Jeffamine D-230 Jeffamine D-400 bis(3-aminopropyl)-terminated PEO (PEG34 diamine)

5 6 7 8

surfactant concentration w/v %

DLS count rate/ kcps

normalized intensity (%)

intensity-average diameter/nm (PDI)

0.0

4.6 × 105

100

218 (0.073)

× × × × × × × × × × × × × ×

19 0.1 0.2 0.6 94.5 3.7 0.03 0.07 90 79 0.4 0.8 1.4 1.45

178 808 682 740 220 125 116 10 302 523 14 19 25 34

0.1 0.50 1.0 5.0 0.10 0.50 1.0 5.0 5.0 10.0 0.10 0.35 0.60 2.2

8.8 3.8 7.5 2.8 4.35 1.7 1.6 3.4 4.15 3.6 2.0 3.6 6.6 6.7

1200

104 102 102 103 105 104 102 102 105 105 103 103 103 103

(0.065) (0.812) (0.766) (0.885) (0.047) (0.085) (0.250) (0.427) (0.118) (0.089) (0.24) (0.21) (0.18) (0.36)

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Table 2. Effect of Adding Ionic Surfactant to 1.0 w/v % Aqueous Dispersions of Both Linear and Crosslinked PGMA55(PHPMA247-stat-PGlyMA82) Copolymer Vesicles at 20 °C entry

type of added crosslinker

type of added surfactant

surfactant concentration w/v %

1 2 3 4 5 6 7 8 9 10 11 12

none none none none ethylenediamine ethylenediamine Jeffamine D-230 Jeffamine D-230 Jeffamine D-400 Jeffamine D-400 PEG34 diamine PEG34 diamine

none anionic cationic cationic none anionic none anionic none anionic none anionic

0.0 1.0 1.0 2.0

DLS derived count rate (kcps)

normalized intensity (%)

× × × × × × × × × × × ×

100 0.2 5.3 0.2 74 83 96 94 85 87 81 80

4.7 1.0 2.5 1.0 3.5 3.9 4.5 4.4 4.0 4.1 3.8 3.75

1.0 1.0 1.0 1.0

105 103 104 103 105 105 105 105 105 105 105 105

intensity-average diameter/nm (PDI) 225 26 150 24 301 338 284 326 281 328 385 414

(0.101) (0.787) (0.267) (0.472) (0.155) (0.089) (0.134) (0.143) (0.125) (0.146) (0.106) (0.109)

workers.66 The same PGMA55 macro-CTA was used for the statistical copolymerization of HPMA and GlyMA. This protocol differs from that used to synthesize PGMA55PHPMA330 by the incorporation of 25 mol % of GlyMA units within the second membrane-forming block. It is well-known that the epoxy group of glycidyl methacrylate is susceptible to ring-opening in the presence of water to form glycerol monomethacrylate.75 However, this requires an acid catalyst, which is not present in our formulation. Alternatively, the epoxy groups could potentially react with the alcohol functionality on the HPMA (or GMA) repeat units. Thus it is worth asking whether the epoxy groups actually remain intact after the copolymer synthesis. Accordingly, the final PGMA55-P(HPMA247-stat-GlyMA82) copolymer was immediately isolated by freeze-drying and then dissolved in d6-DMSO to quantify its epoxy group content by 1H NMR spectroscopy. Epoxy proton signals are clearly visible at 2.6 and 2.8 ppm (see Figure 2A). Comparison of these integrated signals with those due to the methacrylic backbone suggests that at least 90% of the original epoxy groups remained intact after the statistical copolymerization of HPMA with GlyMA after 4 h at 70 °C at pH 4−5. However, GPC analysis of this PGMA55-P(HPMA247-stat-GlyMA82) copolymer indicates a significant increase in its degree of branching under these reaction conditions compared to that of the corresponding linear PGMA55-PHPMA330 diblock (see Figure 2B). This is most likely due to a minor fraction of the epoxy groups reacting with the HPMA (or GMA) repeat units via their alcohol functionality. The resulting copolymer vesicles were characterized by DLS and TEM (see entry 1 in Table 2 and Figure 4). These epoxyfunctionalized copolymer vesicles can react rapidly with nucleophilic compounds such as primary amines in aqueous solution under mild conditions.73,76 A schematic representation of this cross-linking reaction is shown in Figure 1. This epoxyamine chemistry has been widely studied over the last fifty years in the context of the development of epoxy resins.77,78 The reaction is actually rather more complex than that depicted in Figure 1, because secondary amines exhibit similar reactivity toward epoxy groups as primary amines.79 Hence in principle one primary amine may actually react with two epoxy groups. There is also the theoretical possibility of reaction between epoxy groups and the secondary hydroxyl groups on the HPMA units, although this usually proceeds much slower than the epoxy-amine reaction. Such secondary reactions generally lead

These studies revealed the presence of rather ill-defined aggregates of “nano-objects” of around 20−100 nm when treated with either ionic surfactant, confirming loss of the vesicular morphology. In contrast, well-defined vesicles of comparable size to those present originally could still be observed after addition of neutral surfactant. We have recently found that the nano-objects formed by RAFT polymerization-induced self-assembly in aqueous solution are rather sensitive to the incorporation of relatively low levels of ionic comonomers.74 Spheres can still be obtained, but it is much harder to obtain higher order morphologies such as vesicles. Considering these findings in the context of the present work, the introduction of charge via ionic surfactant binding to the copolymer chains presumably causes dissociation of the rather delicate vesicular nanostructures in favor of spheres by a similar mechanism. In contrast, binding of the non-ionic surfactant is much less disruptive to the copolymer vesicles. The apparently greater disruptive power of the anionic surfactant compared to the cationic surfactant observed herein is most likely simply due to the former’s lower molecular weight (hence its greater molar concentration for a given mass concentration). It is also worth emphasizing that these linear copolymer vesicles were also either completely or partially destroyed after addition of either ethylenediamine or various polymeric diamines (see entries 5−10, Table 1). This can be attributed to the amphiphilic character of these additives (which can behave as pseudosurfactants) and/or to their intrinsic basic character (e.g., a 0.35 w/v % aqueous solution of Jeffamine D230 has a solution pH about 11). In summary, the observations reported in Table 1 confirm that the self-assembled linear PGMA55-PHPMA330 diblock copolymer vesicles are readily disrupted by the addition of either ionic surfactants or diamines, but are relatively tolerant of the non-ionic surfactant. Synthesis of Cross-Linked Diblock Copolymer Vesicles. Figure 1 shows a schematic representation of the cross-linking strategy used in this study. The RAFT synthesis of linear (noncross-linked) PGMA55-P(HPMA247-stat-GlyMA82) copolymer vesicles was also conducted at 70 °C (see the Supporting Information). Although the reaction solution is not transparent at 20 °C, it becomes a clear single-phase system within 10 min at 70 °C. Hence we believe that this copolymer synthesis proceeds under aqueous dispersion polymerization conditions,67,68 rather than the aqueous emulsion polymerization conditions previously reported by Charleux and co1201

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Figure 4. Transmission electron micrographs obtained for (a) noncross-linked PGMA55-P(HPMA247-stat-GlyMa82) copolymer vesicles and the same vesicles after epoxy-amine cross-linking using (b) Jeffamine D-230, (c) Jeffamine D-400, or (d) PEG34 diamine.

P(HPMA247-stat-GlyMA82) vesicles at 20 °C (see Table 2). The primary amine/epoxy molar ratio used throughout this series of experiments was 2.0 (i.e., [diamine]/[epoxy] = 1.0). The first entry indicates the original intensity-average diameter (225 nm) and polydispersity (0.101) of these vesicles, as judged by DLS. The initial scattered light intensity is also stated for this reference sample: this value is used to normalize the light intensities obtained after addition of cross-linker and/or surfactant to the vesicles. Given that these diblock copolymer vesicles can be readily prepared as relatively concentrated aqueous dispersions, it seemed pertinent to investigate cross-linking at 10% solids, as well as in dilute solution. PGMA-P(HPMA-stat-GlyMA) copolymer vesicles could be covalently stabilized with Jeffamine D-230 using a [diamine]/[epoxy] molar ratio of 1.0. In contrast, employing Jeffamine D-400 under the same

to higher degrees of cross-linking. However, for the sake of clarity, only reaction of primary amines with epoxy groups is shown in Figure 1. We chose to compare the performance of a small molecule cross-linker (ethylene diamine) with that of several cheap commercially available polymeric diamines (Jeffamines) of varying molecular weight (see Figure 2). A similar cross-linking strategy has been recently reported by Chen and co-workers, who produced a range of covalently stabilized “nano-objects” (vesicles, bilayers, cylindrical micelles and spheres).63,72 However, in their case vesicles were prepared via a post-polymerization processing route, rather than by polymerization-induced self-assembly. Moreover, the GlyMA units were present as a pure block, rather than statistically distributed along a non-reactive polymer chain. In the present study, various diamines were mixed in turn with identical 1.0 w/v % aqueous dispersions of PGMA551202

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entries 6, 8, 10, and 12 in Table 2). This may be due to subtle changes in morphology, such as membrane swelling caused by intercalated surfactant molecules and/or a change in refractive index of these nano-objects. Morphological Changes during Diamine Cross-Linking. TEM studies were undertaken for selected PGMA55P(HPMA247-stat-GlyMA82) copolymer vesicles; Figure 4 depicts representative images obtained before and after diamine cross-linking. Clearly, the addition of diamine has a dramatic influence on the particle morphology: the surface of the original linear vesicles is relatively smooth and featureless, whereas cross-linked vesicles exhibit much greater surface texture. In principle, this intriguing morphology may be the result of competition between diamine-induced vesicle degradation and the covalent stabilization amine/epoxy reaction. Thus the surfactant/basic character of the diamine additive tends to break up the vesicles into nanospheres but the kinetics of crosslinking is sufficiently rapid to produce a hybrid nanostructure: a vesicle membrane comprising cross-linked nanospheres. However, quite similar changes in morphology can be achieved when cross-linking block copolymer nanoparticles prepared via aqueous dispersion polymerization using ethylene glycol dimethacrylate as a comonomer.80 Hence an alternative explanation may simply be that cross-linking induces nanophase separation within the vesicle membrane. Cationic Character of Cross-Linked Copolymer Vesicles. Diamine-cross-linked copolymer vesicles were first purified by dialysis against water (cut-off = 50,000 Da) to remove any excess diamine that did not react with the epoxy groups. Table 3 shows the nitrogen microanalytical data

conditions led to substantial agglomeration. However, by reducing the [diamine]/[epoxy] molar ratio to 0.50, successful vesicle cross-linking was achieved at 10 w/v % using all four diamine cross-linkers. DLS studies indicated that copolymer vesicles cross-linked at 10 w/v % had very similar diameters to those cross-linked at 1.0 w/v %, which suggests that vesicle fusion is negligible under the former conditions (see the Supporting Information). This interpretation is supported by TEM studies (see the Supporting Information). We also briefly investigated cross-linking using a higher molecular weight Jeffamine (D-2000). However, this reagent exhibited inverse temperature solubility behavior at around 20 °C and attempted cross-linking at 10% solids led to visible precipitation even at a [diamine]/[epoxy] molar ratio of 0.50. Surfactant Resistance of Diamine-Cross-Linked Copolymer Vesicles. First, the vulnerability of linear PGMA55P(HPMA247-stat-GlyMA82) copolymer vesicles toward an ionic surfactant challenge was examined (Table 2, see entries 2−4). In all cases, vesicle dissolution was observed within minutes at 20 °C, as judged by visual inspection and subsequent DLS studies. Thus these control experiments indicate that the partial loss of epoxy groups indicated by NMR (see Figure 2A) merely leads to branching after 4 h at 70 °C, with little or no intrinsic cross-linking, since the final copolymer remains sufficiently soluble for GPC analysis (see Figure 2B). In the absence of any diamine cross-linker, the membranes of these epoxy-functional copolymer vesicles are rapidly disrupted by the addition of 1−2% ionic surfactant (see entries 2−4 in Table 2), just like the PGMA-PHPMA diblock copolymer vesicles reported in Table 1. The linear copolymer vesicles proved to be particularly intolerant of the presence of anionic surfactant. Hence this additive was chosen to verify the surfactant resistance of the cross-linked vesicles. Given that the final size observed by DLS after such a surfactant challenge is 24−26 nm, this suggests that the vesicles dissociate to form spherical micelles, rather than molecularly dissolved copolymer chains. Addition of diamine cross-linker causes a modest increase in PGMA55-P(HPMA247-stat-GlyMA82) vesicle diameter (see entries 5, 7, and 9 in Table 2), but these diaminetreated vesicles appear to be covalently stabilized since they are colloidally stable in the presence of ionic surfactant (see entries 6, 8, and 10 in Table 2). Nevertheless, there is still some reduction in light scattering intensity, which may indicate the extraction of a minor soluble fraction of copolymer chains, or perhaps partial break-up into smaller micelles. Unlike the linear PGMA55-PHPMA330 copolymer vesicles, the addition of diamine to linear PGMA55-P(HPMA247-statGlyMA82) vesicles does not cause a dramatic reduction in either their colloidal dimensions or the corresponding normalized light scattering intensities (compare entries 5−8 in Table 1 with entries 5, 7, 9, and 11 in Table 2). In all cases, DLS confirmed that comparable sizes and light scattering intensities were observed before and after cross-linking. Given that the diamines proved to be disruptive toward the linear PGMA55-PHPMA330 copolymer vesicles, this suggests that the kinetics of crosslinking of the PGMA55-P(HPMA247-stat-GlyMA82) vesicles must be faster than the diamine-induced degradation of the copolymer vesicles. It is perhaps also noteworthy that DLS studies indicate an increase in intensity-average diameter (and polydispersity) after diamine cross-linking (compare entry 1 with entries 5, 7, 9, and 11 in Table 2). A further modest size increase is observed on addition of ionic surfactant to the cross-linked vesicles (see

Table 3. Summary of Nitrogen Microanalyses Obtained for Linear and Jeffamine 230 Cross-Linked PGMA55P(HPMA247-stat-GlyMA82) Copolymer Vesiclesa

linear PGMA55-P(HPMA247-stat-GlyMA82) cross-linked PGMA55-P(HPMA247-statGlyMA82) as-synthesized after dialysis after centrifugation

N% (theory)

N % (expt)

0.025

not detected

3.20 3.20 3.20

2.50 0.80 0.90

a

In each case, crosslinking was conducted at 1.0 w/v % copolymer concentration using one equivalent Jeffamine D-230 based on GlyMA groups.

obtained for linear and cross-linked copolymer vesicles purified by either dialysis or repeated centrifugation. As expected, the original linear PGMA55-P(HPMA247-stat-GlyMA82) copolymer vesicles contained no detectable nitrogen, since that due to the RAFT CTA corresponds to less than 0.03%, which is below the detection limit of 0.20%. The actual nitrogen content of the crude PGMA55-P(HPMA247-stat-GlyMA82) copolymer crosslinked with Jeffamine D-230 was reasonably close to that expected based on the reaction stoichiometry (2.50 vs 3.20%). However, after dialysis the copolymer nitrogen content was approximately three-fold lower (0.80%), suggesting that only around one-third of the Jeffamine D-230 cross-linker had actually reacted with the copolymer vesicles. Moreover, a similar nitrogen content (0.90%) was obtained using the centrifugation/redispersion purification protocol (three cycles at 10 000 rpm for 1 h for each cycle). 1203

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the presence of ionic surfactant. Moreover, using such epoxyamine cross-linking chemistry confers cationic character on the copolymer vesicles, since some residual pendent amine groups remain within the vesicles. Finally, cross-linking also leads to a distinctive change in particle morphology, with phase separation occurring on the nanometer length scale within the vesicular membrane.

This diamine cross-linking methodology not only produces unique morphologies and excellent surfactant resistance, but also allows amine groups to be conveniently introduced into the vesicles. These may be unreacted pendent primary amines (if just one primary amine group on the diamine cross-linker reacts with either one or two epoxy groups) or secondary or tertiary amines (if a given primary amine reacts with either one or two epoxy groups, respectively). In all cases, these amine groups are readily protonated at low pH and hence should confer cationic character on the copolymer vesicles. Such amine functionalization is readily confirmed by aqueous electrophoresis studies (see Figure 5) of both linear copolymer



ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectrum of polymerizing reaction solution recorded after 4 h at 70 °C; TEM images for linear diblock copolymer vesicles obtained before and after addition of surfactant; DLS data and TEM images for diamine-cross-linked copolymer vesicles. This material is available free of charge via the Internet at http://pubs.acs.org.

■ ■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]

ACKNOWLEDGMENTS G.B. and S.P.A. thank Yorkshire Forward for partially funding a postdoctoral grant to support P.C. S.P.A. and G.B. also thank EPSRC for a Platform grant (EP/E012949/1) to support A.B.



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Figure 5. Zeta potential vs pH curves obtained for PGMA55PHPMA330 and PGMA55-(PHPMA247-stat-PGlyMA82) copolymer vesicles after cross-linking using Jeffamine 230 and also after purification of these cross-linked vesicles by dialysis.

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CONCLUSIONS Linear methacrylic diblock copolymer vesicles are conveniently synthesized at 10% solids using an aqueous dispersion polymerization formulation. Such vesicles can tolerate the presence of nonionic surfactant, but do not survive exposure to ionic surfactants. Epoxy-functional copolymer vesicles can be readily prepared by incorporating glycidyl methacrylate as a comonomer. These epoxy groups can be readily cross-linked using commercially polymeric diamines in aqueous solution at 10% solids. These cross-linked vesicles are colloidally stable in 1204

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