Broadening the range of vesicle formation by heating

Abstract

Vesicles of amphiphilic block copolymers have been extensively studied, but surprisingly few studies used high temperature to promote the polymer shape evolution towards vesicles. In this work, we carry out a systematic comparison between the popular water addition method and the heating method, and the latter was found to have a much broader window of conditions giving vesicles. For most of the conditions we attempted, it appears that the evolution towards vesicles is largely limited by kinetics, though allowed by thermodynamics. Hence, heating is instrumental in promoting the process. We believe that this demonstration of simple concept would popularize the heating method and help vesicle preparation in this field.

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1. Introduction

Vesicles of amphiphilic block copolymers (BCPs) have been extensively studied, not only to explore the mechanisms of polymer self-assembly, but also to exploit their hollow cavity for imaging,^1–5 drug delivery,^6–12 and other applications.^13–16

Generally, the vesicle-forming BCPs can be classified into two categories, the first of which bear hydrophobic blocks with relatively low glass transition temperatures, such as poly(butadiene)-b-poly(ethylene oxide) (PB-b-PEO). Owing to their high flexibility, these BCPs can easily form vesicles in water under ambient conditions, but it would be equally easy for the resulting vesicles to further transform releasing cargo inside the cavity. The other category consists of copolymers whose hydrophobic blocks have a high glass transition temperature, such as polystyrene-based BCPs. The polystyrene block usually exists as a glassy state under ambient temperature,¹⁷ and this low flexibility leads to slow rates of micellar evolution. But once vesicles form, their toughness and high kinetic stability¹⁸ allow them to survive better in harsh environments. Hence, vesicles made of glassy BCPs have been widely studied^19–22 and applied in various applications.^6,18

In terms of preparation, vesicles of non-glassy copolymers are usually prepared by a direct hydration method,^23–25 sometimes with additional techniques such as electroformation,²⁶ microfluidics,²⁷ etc.²⁸ In contrast, the evolution of glassy copolymers in water is too slow for these methods. Hence, the preparation method always involves organic co-solvents^{20,22,28–31} to swell the polymer domain and assist its shape transformation. In a typical procedure, a BCP is first dissolved in a good solvent such as dimethylformamide (DMF) or tetrahydrofuran (THF), which can dissolve both the hydrophilic and the hydrophobic blocks. Water is then slowly added as the second solvent, which is miscible with the first solvent but does not dissolve the hydrophobic block, forcing the BCPs out of the solution as micelles. As the water content increases in the solution, the BCP micelles first form by polymer aggregation, and then evolve through spherical and cylindrical micelles towards vesicles.

For the water addition method, glassy vesicles can only be obtained in a narrow range of experimental conditions. Typically, low water contents are used to achieve a high degree of swelling; high polymer concentrations are used to facilitate the aggregation of polymers and micelles; and acids or salts are used to further reduce/shield the charge repulsion among the ionic blocks.²¹ Even with optimized conditions, the preparation often requires a long period (12–36 h). Most importantly, the choice of BCPs is limited to those with neutral hydrophilic blocks (e.g., PS-b-PEO) or “crew-cut” BCPs, where the ionic blocks take less than 20% of the total chain length. “Long-hair” ionic BCPs are rarely used for making vesicles.

Previously, we developed a heating assisted method to encapsulate various nanoparticles in BCP shells^32,33 and to prepare toroidal micelles³⁴ and vesicular intermediates.³⁵ Basically, a BCP is dispersed in a solvent mixture (e.g., DMF/water) and then heated at 60–110 °C. The high kinetic energy of the polymer facilitates their aggregation and micellar evolution. In this work, we carry out a systematic comparison between the water addition method and the heating method in terms of preparing vesicles. We show that for crew-cut and neutral BCPs, the heating method can prepare vesicles at a broader range than the popular water addition method: vesicles can be prepared at a lower concentration, higher water content, and much shorter time period. Most importantly, we show that long-hair ionic BCPs could form vesicles, which is not possible using the water addition method.

2. Results and discussion

It is easily conceivable that the higher kinetic energy at a higher temperature would facilitate the various steps of micelle evolution, including polymer aggregation, micelle aggregation, micelle coalescence, and shape transformation. Despite the obvious advantages, we are surprised that very few studies employed elevated temperature when synthesizing vesicles, perhaps also because the field is more occupied with polymer syntheses than exploring new preparative conditions. Without precedents in the literature, it is unclear to what extent heating can facilitate vesicle formation. To the best of our knowledge, there is to date no systematic study comparing the heating method to the water addition method.

2.1 Choice of experimental conditions

In the conventional water addition method (Scheme 1, a), a BCP is first dissolved in a good solvent (DMF or THF). A small amount of water is then slowly added through a syringe pump under vigorous stirring. In our experiments, the flow rate was set at 10 μL min⁻¹ for all samples, until a pre-determined solvent ratio was reached. The mixture was then kept under stirring for additional 12 h to allow the micelles to evolve.

Scheme 1 Schematics showing the two methods of vesicle preparation: (a) the water addition method and (b) the heating method.

In our heating method (Scheme 1, b), the same BCP was dissolved in the same organic solvent. An appropriate amount of water was then directly added to reach the same pre-determined solvent ratio, and the mixture was heated at 110 °C for 4 h to promote micelle evolution. Prolonged heating gave only minor improvements and was thus not adopted due to efficiency considerations.

To compare properly, the two methods were adapted from the literature ones, so that similar conditions were used, for example, the choice of organic solvent and the solvent ratio. Moreover, the post-synthetic treatments were also unified: a large amount of water was added to the reaction mixture after the synthesis, so as to extract the organic solvent from the micelles locking their structure. Due to the glassy nature of the polystyrene-based BCPs, the micelles are kinetically frozen and can be directly dried for characterization by transmission electron microscopy (TEM).^29,36

2.2 Vesicles of crew-cut anionic BCPs

As a model system, we first compare the vesicle formation of a crew-cut anionic BCP (PS₁₉₂-b-PAA₁₃), which has been extensively studied using the water addition method.^{19,20,22,30,37}

First, we compared the dependence on the polymer concentration, with the solvent ratio set as a constant (DMF/H₂O = 10 : 2). It is easily conceivable that, at a higher BCP concentration, the probability of polymer aggregation (among the polymer molecules) and micelle aggregation (among the initially formed micelles of various forms) will be promoted, facilitating the subsequent vesicle formation. As shown in Fig. 1, both methods failed to produce vesicles when BCP concentration was at 0.1 wt%, only giving large spherical structures (Fig. 1, a1 and b1). As the BCP concentration increased progressively, the heating method started to form vesicles as low as 0.2 wt% (with low yield), and the vesicles became larger with higher yield (Fig. 1, a2–a4) with further increase of BCP concentration. At 1.6 wt%, vesicles with multiple compartments were obtained (Fig. 1, a5), likely because of the extensive polymer aggregation at the initial stage.

Fig. 1 Dependence on BCP concentration, using PS₁₉₂-b-PAA₁₃ at a constant solvent ratio (DMF/H₂O = 10 : 2). The products of the heating method: (a1) 0.1 wt%, (a2) 0.2 wt%, (a3) 0.4 wt%, (a4) 0.8 wt%, and (a5) 1.6 wt%. The products of the water addition: (b1) 0.1 wt%, (b2) 0.2 wt%, (b3) 0.4 wt%, (b4) 0.8 wt%, (b5) 1.2 wt%, and (b6) 1.6 wt%. Circle indicates successful vesicle formation, whereas cross indicates failed formation. Partial success is represented by a cross over circle. The same symbols are used in the following images. Scale bar: 100 nm.

In contrast, using the water addition method, no vesicle was obtained even at a high BCP concentration of 0.8 wt%, where the products changed from irregular shaped polymer aggregates to short branched cylinders and lamellae (Fig. 1, b2–b4). As the BCP concentration reached 1.6 wt%, clear vesicular micelles could be observed, but still with signs of incomplete vesicle evolution (Fig. 1, b6). To explore the threshold concentration, an extra point was set at 1.2 wt%, where partially evolved cylinder–lamella–vesicle composites were observed (Fig. 1, b5). Hence, the results show a clear trend that higher BCP concentration promotes vesicle formation.

Next, the BCP concentration was set at a constant value of 0.8 wt%, while the DMF/water ratio was varied. Using the heating method (4 h), vesicles can be obtained at a wide range of solvent ratios (from 10 : 1 to 10 : 6, Fig. 2, a2–a4). The vesicles appear to decrease in size as the water content increases, from hundreds of nm to around 100 nm (10 : 6), as clearly evident from the volume percentage of the vesicular cavities. With extremely low water content (10 : 0.5), only irregularly shaped loose polymer structures were observed (Fig. 2, a1). When the water content was too high (10 : 8), only spherical structures were observed (Fig. 2, a5), likely because the glassy PS domains were kinetically locked without sufficient swelling by the organic solvent.

Fig. 2 Dependence on the water content, using PS₁₉₂-b-PAA₁₃ at a constant BCP concentration (0.8 wt%). The products of the heating method: (a1) DMF/H₂O = 10 : 0.5, (a2) 10 : 1, (a3) 10 : 4, (a4) 10 : 6, and (a5) 10 : 8. The products of the water addition: (b1) DMF/H₂O = 10 : 0.5, (b2) 10 : 1, (b3) 10 : 4, and (b4) 10 : 8. Scale bar: 100 nm.

In contrast, the water addition method (12 h) gave only a narrow window of vesicle formation. Only at the ratio of 10 : 1, multi-compartment vesicles were obtained (Fig. 2, b2). Other than the small window of success, the overall trend is similar as that of the heating method: lower water content gave loose polymer aggregates (Fig. 2, b1); whereas higher water content gave significant amounts of cylindrical and lamellar components (Fig. 2, b3 and b4) indicating extremely slow evolution process.

The dependence of vesicle formation on both BCP concentration and solvent ratio was summarized in Fig. 3, with a few added points (Fig. S1†) to help delineate the rough range. For both methods, the overall trends are similar to those in the literature¹⁷ in that vesicles are more favoured at the upper right corner (high BCP concentration and high water content), and that above a certain water content, the micelle evolution starts to be limited by the rate of transformation. The direct comparison of the two plots clearly shows that the heating method can obtain vesicles at a much broader range than the water addition method. Even with shorter time (4 vs. 12 h), the heating method leads to more extensive micelle evolution.

Fig. 3 Summary showing the window of vesicle formation for the crew-cut BCP, PS₁₉₂-b-PAA₁₃: (a) the heating method; and (b) the water addition method.

2.3 Mechanistic interpretation

For both methods, once the BCPs are excluded from the solvent, they start to aggregate, from small molecular clusters, to spherical micelles, cylindrical micelles, and lamellar micelles. Their shape evolves concurrently, eventually leading to vesicles³⁵ with enclosed cavity. The thermodynamic driving force is the tendency to reduce the surface energy, which is reflected in the packing parameter¹⁷ or the surface/volume ratio of the hydrophobic domain.³⁸ In other words, the larger the structure is, the more stable it becomes. On the other hand, the steric and charge repulsion of the hydrophilic blocks favors small structure with higher curvatures. These two factors, along with the entropic term of the hydrophobic domain (it cannot be thicker than the physical length of the coiled hydrophobic block), determine the thermodynamic end-point of micelle evolution (sphere, cylinder, or vesicle). Detailed discussion can be found in reviews.^17,29,30,38 As such, the different temperatures of the two methods are not expected to determine the thermodynamic end-points.

In contrast, the kinetic aspects are critically dependent on the temperature, including the rates of polymer aggregation, of micelle aggregation, of micelle coalescence, and of shape transformation. While these processes are often discussed as a combined one, it is clear that they are distinct in nature with drastic differences of reaction intermediate and activation energy. In general, the evolution towards the thermodynamic end-points is limited by BCP concentration,³⁹ water content,^39–41 temperature,³⁴ time, and the intrinsic property of BCPs.^34,35 Basically, the BCP concentration affects the probability of collision among the monomers, small clusters, and micelles. While such collisions are obviously very complex, a large BCP concentration helps the rates in general. The solvent quality (i.e., water content) affects both the thermodynamic end-point and the micelle evolution kinetics. At low water content, the PS domains are highly swollen by the organic solvent, facilitating the movement and rearrangement of the polymer chains. Hence, for high water content and low BCP concentration conditions, micelle evolution is expected to be slow and heating is particularly important.

While it is easily conceivable that a higher temperature would promote all kinetic processes (collisions, rearrangements, etc.), the extent of benefits remains unclear. Our data clearly shows the advantage. Most literature reports on vesicle preparation used room temperature, which may lead to false negatives. Indeed, Fig. 3 demonstrates that a large area of the conventional “phase diagram” contains false negatives—vesicles are thermodynamically feasible but cannot form due to extremely slow rates of evolution.

In our previous studies of encapsulating nanoparticles with BCPs,⁴² heating was always applied and it appears to be easier with a broader scope than the water addition method,⁴³ likely by facilitating the polymer self-assembly.⁴⁴ In these systems, the polymers adsorb on hydrophobically functionalized nanoparticle surface and hence, the BCP structure is similar to that of spherical micelles, rather than the vesicles of this study.

2.4 Vesicles of cationic and neutral BCPs

To extend the method comparison to other types of BCPs, we chose a crew-cut cationic BCP, PS₂₂₂-b-P4VP₄₃ and a neutral BCP, PS₁₄₄₂-b-PEO₇₉₅, both of which have been studied using the water addition method.^22,30,37,45

For PS₂₂₂-b-P4VP₄₃, the solvent ratio was set at THF/H₂O = 10 : 3 and the BCP concentration was varied. In the literature, vesicles of PS-b-P4VP were synthesized via the water addition method, either in DMF with additional ions⁴⁵ or in less polar solvents such as THF and dioxane.³⁰ We tried to avoid salts and chose THF as solvent to make our comparison more relevant to the literature.

Using the heating method, vesicles could be obtained under a wide range of BCP concentrations from 0.2–1.6 wt% (Fig. 4, a1–a4). At 0.2 wt%, vesicles were found along with cylindrical structures (Fig. 4, a1), indicating incomplete evolution process. Such intermediates disappeared with increase of BCP concentration. In the water addition method, however, vesicles were obtained when BCP concentration was 0.8 wt% or higher (Fig. 4, b3 and b4). At lower concentrations of 0.4 and 0.2 wt% (Fig. 4, b1 and b2), short cylindrical segments were observed. The direct comparison shows that the extent of evolution was slower in the water addition method.

Fig. 4 Dependence on BCP concentration. The heating method (90 °C for 4 h) for PS₂₂₂-b-P4VP₄₃ at THF/H₂O = 10 : 3: (a1) 0.2 wt%; (a2) 0.4 wt%; (a3) 0.8 wt%; and (a4) 1.6 wt%; and the water addition method: (b1) 0.2 wt%; (b2) 0.4 wt%; (b3) 0.8 wt%; and (b4) 1.6 wt%. The heating method (110 °C for 4 h) for PS₁₄₄₂-b-PEO₇₉₅ at DMF/H₂O = 10 : 1: (c1) 0.2 wt%; (c2) 0.4 wt%; (c3) 0.8 wt%; and (c4) 1.6 wt%; and the water addition method: (d1) 0.2 wt%; (d2) 0.4 wt%; (d3) 0.8 wt%; and (d4) 1.6 wt%. Scale bar: 100 nm.

We chose DMF as solvent for PS-b-PEO in order to be consistent with the water addition method in the literature.^21,37 At a constant solvent ratio (DMF/H₂O = 10 : 1), similar conclusions as above were obtained for PS₁₄₄₂-b-PEO₇₉₅: the heating method successfully induced vesicle formation within a large window of BCP concentration from 0.2–1.6 wt% (Fig. 4, c1–c4), with multi-compartment vesicles formed at higher concentrations. On the other hand, the water addition method could only produce vesicles at the highest BCP concentration of 1.6 wt% (Fig. 4, d4), with the other three samples giving solid spherical structures (Fig. 4, d1–d3).

2.5 Vesicles of long-hair ionic BCPs

In the literature, glassy vesicles were synthesized from either crew-cut ionic BCPs^20,30,37 or BCPs with neutral hydrophilic blocks,^20,37 whereas attempts of using “long hair” ionic BCPs were never successful for the water addition method (without additives). We extended the comparison to long-hair ionic BCPs, as it is conceivable that the higher kinetic energy at higher temperature would also promote their evolution. Since there is no literature precedent to follow in terms of feasible preparative conditions, we have to search for optimal conditions and carry out comparison there.

With longer hydrophilic blocks, it is expected that the window for vesicle formation would be even smaller: from the thermodynamic point of view, the repulsion among the hydrophilic blocks on the surface of a micelle is expected to be stronger, making it less favorable to form vesicles.³⁵ To counter act, higher water content would be necessary to increase the polymer–solvent interfacial energy and thus favor vesicles. From the kinetic point of view, however, higher water content would cause even slower rate of micellar evolution, making vesicles kinetically inaccessible.

With these considerations, we screened the preparative conditions using the heating method, where the time period was often extended to 6–10 h to ensure sufficient time for the polymer evolution. Once vesicles can be obtained, it will prove the thermodynamic feasibility. Then, the water addition method was carried out using the same optimal conditions.

A tri-block cationic BCP, P4VP₄₃-b-PS₂₆₀-b-P4VP₄₃, was selected. It has two P4VP blocks and is thus expected to evolve slower than the above diblock BCP with similar length of hydrophilic blocks, PS₂₂₂-b-P4VP₄₃. The comparison was first performed at a constant solvent ratio of DMF/H₂O = 10 : 2 with varying BCP concentration. With the heating method (110 °C for 4 h), vesicles can be obtained at BCP concentrations of 0.8 and 1.6 wt% (Fig. 5, a3 and a4). At 0.4 wt%, vesicles can be found but with very low yield (Fig. 5, a2) and no vesicles were observed at 0.2 wt% (Fig. 5, a1). Applying the same conditions to the water addition method gave only partially linked spherical micelles in all cases (Fig. 5, b1–b4). No smooth cylinders could be found, not to mention well developed vesicles. Such outcomes are consistent with the literature findings and clearly show the advantage of the heating method.

Fig. 5 Vesicle formation from a long-hair triblock BCP, P4VP₄₃-b-PS₂₆₀-b-P4VP₄₃. In DMF/H₂O = 10 : 2, applying the heating method (110 °C for 4 h): (a1) 0.2 wt%; (a2) 0.4 wt%; (a3) 0.8 wt%; and (a4) 1.6 wt%; and the water addition method: (b1) 0.2 wt%; (b2) 0.4 wt%; (b3) 0.8 wt%; and (b4) 1.6 wt%. At 1.6 wt%, applying the heating method: (c1) THF/H₂O = 10 : 3, (c2) 10 : 6, (c3) 10 : 9, and (c4) 10 : 12; and the water addition method: (d3) THF/H₂O = 10 : 9 and (d4) 10 : 12. Scale bar: 100 nm.

A separate study was carried out using a BCP concentration of 1.6 wt% and screening the optimal THF/H₂O solvent ratio (in contrast to the DMF/H₂O mixture above). With the heating method (90 °C for 6 h), clean vesicles were obtained at 10 : 12 ratio (Fig. 5, c4); a mixture of cylinders and vesicles were obtained at 10 : 9 ratio (Fig. 5, c3); and no vesicles could be found at lower water contents (Fig. 5, c1 and c2). In general, the cylinders and vesicles obtain in THF/H₂O mixtures were smoother than those obtained in DMF/H₂O mixtures, likely because the lower polarity of THF can better swell the PS domains and facilitate structural evolution. Using the best solvent ratio, a high BCP concentration, and THF as the solvent, these optimal conditions were then applied in the water addition method: only a few vesicles were observed at the solvent ratio of 10 : 9 (Fig. 5, d3), but not at all at 10 : 12 (Fig. 5, d4).

Next, we chose a negatively charged long-hair BCP, PS₁₅₄-b-PAA₄₉ (PAA% = 24.1%), whose hydrophilic blocks are relatively longer than the crew-cut BCPs^20,30,37 in the literature (PAA% < 10%). At BCP concentration = 2 wt% and without additives, initial screening of DMF/H₂O solvent ratio using the heating method (110 °C for 10 h) gave no vesicles. It is known that acid^21,34,35 and oligoamine^28,41,46,47 can suppress the charge on the PAA blocks and favors vesicle formation. On the other extreme, however, too much HCl (200 mM) causes precipitation of the BCP. Even with suitable acid present, there is also a window for water content: too high water content would limit the kinetic evolution of the micelles, whereas at too low water content, vesicles are simply not thermodynamically feasible. Thus, we carried out a rough screening using the heating method (110 °C for 10 h) at intermediate acid concentrations (50 and 100 mM) and solvent ratios (DMF/H₂O = 10 : 1 and 10 : 2). With the lower water content (DMF/H₂O = 10 : 1), the BCP is expected to evolve faster but still no vesicle was obtained (likely thermodynamically unfavorable), only giving spheres at 50 mM HCl (Fig. 6, a1) and irregular-shaped structures at 100 mM HCl (Fig. 6, a2). With increased water content (10 : 2), vesicles were obtained at 100 mM HCl (Fig. 6, a4), whereas the sample at 50 mM HCl produced incomplete vesicles along with cylindrical micelles (Fig. 6, a3). On the basis of these results, the optimized conditions (DMF/H₂O = 10 : 2, [H⁺] = 100 mM) were applied to the water addition method. No vesicle was obtained and the chain-like aggregates of spheres indicate extremely slow evolution (Fig. 6, b4).

Fig. 6 Comparison using long-hair ionic BCPs. For the negatively charged PS₁₅₄-b-PAA₄₉, at 2 wt%, the heating method was applied at 110 °C for 10 h: (a1) DMF/H₂O = 10 : 1, [H⁺] = 50 mM; (a2) 10 : 1 and 100 mM; (a3) 10 : 2 and 50 mM; and (a4) 10 : 2 and 100 mM; the optimal conditions were applied to the water addition method: (b4) DMF/H₂O = 10 : 2, [H⁺] = 100 mM. For the positively charged PS₂₃₀-b-P4VP₉₀, at 2 wt%, the heating method was applied at 90 °C for 10 h: (c1) THF/H₂O = 10 : 3; (c2) 10 : 10; and (c3) 10 : 16; the optimal conditions were applied to the water addition method: (d3) THF/H₂O = 10 : 16. Scale bar: 100 nm.

Generally, long hydrophilic blocks are expected to cause high activation barriers, leading to slow micelle evolution. Once the charges on the hydrophilic blocks are partially neutralized, either by protonation or with oligoamine counter ions, they become more compatible with the hydrophobic domain and can get disentangled more easily during the micelle coalescence and shape evolution. For such difficult situations, heating becomes a critical factor, and hence, prolonged heating (10 h) often makes a difference.

For the positively charged diblock BCP, we chose PS₂₃₀-b-P4VP₉₀ (P4VP% = 28.2%), whose hydrophilic blocks are longer than those used in the literature (P4VP% < 16%).^30,45 With the heating method (90 °C in THF/H₂O for 10 h) and BCP concentration of 2 wt%, initial screening showed that low water content solution did not work for this polymer. For example, THF/H₂O = 10 : 3 (as used in Fig. 4) gave only spherical micelles (Fig. 6, c1); and THF/H₂O = 10 : 10 gave a mixture of cylinders and spheres (Fig. 6, c2). Only when the water content was high enough (10 : 16) that vesicles were obtained along with spheres and cylinders, suggesting that the evolution was still incomplete (Fig. 6, c3). When the water content was too high (10 : 25), however, bulk precipitates were obtained as a result of polymer aggregation. With these information, the optimal condition (THF/H₂O = 10 : 16) was again applied to the water addition method. But still no vesicle was obtained (Fig. 6, d3), likely because the BCP is kinetically frozen at such a high water content.

3. Conclusions

We systematically compared the heating method to the water addition method in terms of vesicle preparation from glassy BCPs. The general trends are consistent with those in the literature that vesicles are favoured at high BCP concentration and intermediate water content. Importantly, heating can greatly extend the window of suitable conditions, not only in terms of BCP concentration and water content, but also for long-hair ionic BCPs which is otherwise inapplicable. The direct comparison demonstrates the inter-connected arguments of thermodynamics versus kinetics:^17,35,38 when the water content is too low (e.g., Fig. 5, c1–2 and 6, c1–2), the kinetic evolution is not a bottleneck but the thermodynamics is unfavourable; but for most of the conditions in Fig. 3, the vesicles are thermodynamically feasible but kinetically inaccessible at room temperature. Without sufficient evolution at high enough temperature, the obtained “phase diagram” of micellar evolution would contain many false negatives. Hence, we advocate that elevated temperature should be generally applied in vesicle preparation, in order to reduce false negative and improve the yield of vesicles.

Acknowledgements

This work was supported by the MOE of Singapore (RG14/13).

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Footnote

† Electronic supplementary information (ESI) available: Experimental section; complementary and large-area view of TEM images (Fig. S1–S60). See DOI: 10.1039/c6ra19913e

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