Straightforward Synthesis Route to Polymersomes with Simple

Letter pubs.acs.org/Langmuir

Straightforward Synthesis Route to Polymersomes with Simple Molecules as Precursors Jan K. Szymański*,† and Juan Pérez-Mercader*,†,‡ †

Department of Earth and Planetary Sciences, Harvard University, Cambridge, Massachusetts, United States Santa Fe Institute, Santa Fe, New Mexico, United States

‡

S Supporting Information *

ABSTRACT: We demonstrate an easy-to-implement experimental emulsion polymerization protocol whose outcome is an amphiphilic copolymer capable of forming vesicles in an aqueous phase. The protocol does not require prior purification of chemicals or the exclusion of oxygen. Using n-butyl acrylate as the monomer, we employ a redox initiation system composed of cerium(IV) ions and poly(ethylene glycol) (PEG), optimizing the performance of this redox couple such that the reaction can be conducted in air. The PEG-based chain radicals produced during initiation attack the monomer molecules, resulting in an amphiphilic product, which brings the synthesis of a vesicle-forming polymer to a level where no complicated equipment is required and may have implications for origins of life research.

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INTRODUCTION Amphiphilic copolymers are a versatile class of diblock copolymers that have been attracting the interest of the community because of their physicochemical properties.1 Among them, AB diblock copolymers, where block A is hydrophilic and B hydrophobic, are particularly interesting because their molecules, in an appropriate liquid medium, can spontaneously form bilayer vesicles. These structures, known as polymersomes, 2,3 are finding uses and applications in biomimetics, pharmaceuticals,4 and, more recently, in trying to address some basic problems in the field of the origins of life.5 Their increased ability to withstand harsh chemical conditions (as compared to the phospholipid-based liposomes) can be made use of in the formulation of proof-of-concept theories and scenarios about the general nature of chemical processes that may have led to the emergence of the first living entities on earth. Because of their similarity in size to extant cells, so-called giant vesicles with diameters of between 1 and 100 μm are particularly relevant in these studies. Polymersomes are one of various self-assembled configurations that can be adopted by amphiphilic copolymers,6 with the exact structures formed under given experimental conditions depending on the requirements that must be met for reaching the necessary energy minimum. This, of course, is directly related to the physicochemical characteristics of the polymeric chains and, most importantly, to the ratio of the mass of the hydrophilic fragment to the total mass of the chain.7,8 Thus, when attempting to synthesize amphiphilic block copolymers for self-assembly purposes, one has to take into consideration that the number of units present in the resulting chain will determine the self-assembly configuration of the product.9,10 It is for this reason that amphiphilic block © 2014 American Chemical Society

copolymers are usually accessed by specialized polymerization techniques.11−13 However, these techniques have drawbacks of extreme sensitivity to impurities such as atmospheric oxygen and moisture and the requirement for specialized initiators and chain-transfer agents, which makes the synthesis rather delicate and difficult to achieve except under ideal laboratory conditions. As a result of the above, recent years have seen increased interest in amphiphilic copolymers prepared without such stringent environmental controls. In addition to hyperbranched polymers,14 there are also reports of vesicles formed from random copolymers. 15,16 Even though both classes of substances lack the well-defined properties of amphiphilic diblock copolymers, the formation of hydrogen bonds and van der Waals interactions allows for the spatial separation of hydrophobic and hydrophilic domains in their chain aggregates and, therefore, for the formation of polymersomes. In this letter, we report on experiments carried out in order to develop and implement a simple synthesis route to amphiphilic copolymers that would require neither ultra-highpurity reagents nor the exclusion of oxygen. Our approach is based on redox-initiated free radical polymerization, an easy-toimplement technique involving the formation of polymerization-initiating radicals through an oxidation−reduction process.17 This methodology has been successfully applied in the synthesis of block copolymers18,19 whereby one of the reactions exploited was the oxidation of an organic component, such as an alcohol, with a metal cation.20,21 The key premise Received: July 16, 2014 Revised: September 8, 2014 Published: September 15, 2014 11267

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Figure 1. (a) Changes in absorbance at 610 nm wavelength over the course of emulsion polymerization of n-butyl acrylate initiated by cerium(IV) and poly(ethylene glycol) for two different volume ratios (R) of monomer to the aqueous phase. (b) Polymer latex absorbance at 610 nm after 3 h of reaction time for different initial ratios (v/v) of acrylate and the aqueous phase. (c) Particle diameter distribution for the final latex obtained for the case of R = 0.172 as measured by DLS. (d) Masses of the final reaction products for different initial ratios (v/v) of acrylate and the aqueous phase. the absorbance at a 610 nm wavelength. The light source was a warm white LED lamp (Thorlabs). The dynamic light scattering (DLS) measurements for size determination were conducted on a Beckman Coulter DelsaNano C machine, and the 1H NMR spectra were acquired in chloroform-d at 25 °C on a 500 MHz Varian Unity/Inova spectrometer. A detailed description of the materials used is given in the Supporting Information. Separation and Purification of the Final Product. Polymerization was allowed to run for 3 h, and as it progressed, the lightorange color of cerium(IV) gradually faded away and the whole mixture became opaque and milky as the polymer latex particles accumulated. After 3 h, samples of the final latex emulsions were subjected to DLS analysis and the bulk of the emulsion was coagulated by stirring in 0.2 g portions of sodium chloride until the solution below the coagulum became clear. The coagulum was filtered off, reprecipitated from acetone by adding deionized water, washed with another portion of water, and air dried. The solid mass was weighed, and the conversion of the monomer was determined gravimetrically and with the aid of 1H NMR. Testing the Product for Vesicle Formation. To test for the ability to form vesicles, the dry final product of the synthesis was subjected to the inverted emulsion transfer method,24,25 one of many giant vesicle formation protocols described in the literature.26 The details are given in the Supporting Information.

here was that the alcohol undergoing oxidation was itself a polymer, in this case poly(ethylene glycol) (PEG),22,23 and therefore that the radicals formed were chain radicals; these were then able to attack a hydrophobic monomer and initiate a reaction sequence that eventually leads to an amphiphilic polymer with fairly well-controlled chain lengths. We show that even though the tight control over the product is lacking, emulsion polymerization of n-butyl acrylate initiated by a redox reaction of PEG with cerium(IV) cations furnishes an amphiphilic product with interesting self-assembly capabilities into vesicles.

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EXPERIMENTAL SECTION

Procedures. In a typical experiment, 3.37 mL of deionized water (12 mΩ), 0.523 g of poly(ethylene glycol), 0.0262 g of sodium dodecyl sulfate (SDS), and a variable amount of n-butyl acrylate (ranging from 0.1 to 0.6 mL) were placed in a tightly sealed 5 mL glass vial equipped with a small magnetic stirring bar. The emulsion was stirred and heated until the temperature of the contents reached 40 °C, at which point 0.116 mL of the ammonium cerium(IV) sulfate (ACN) solution in sulfuric acid was added. The vial contents were kept at 40 °C for the rest of reaction. The turbidity of the reacting mixture was followed online with a fiber optics detector connected to a USB spectrophotometer (Ocean Optics USB 2000+) by measuring 11268

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RESULTS AND DISCUSSION Methodology. We first investigated the polymerization process itself by following the properties of the emulsion as the reaction progressed for different initial amounts of n-butyl acrylate and therefore different volume ratios (R) of the monomer and aqueous phases. To counter the influence of oxygen, the starting poly(ethylene glycol) concentration was larger than in similar experiments employing (N-isopropyl)acrylamide as the monomer19 ([Ce(IV]0/[PEG]0 = 0.07 in our case). We used absorbance measurements at 610 nm as a means to determine the turbidity and thus estimate the solid content of the reacting polymerization mixture. The aforementioned wavelength was selected because of the global absorbance minimum exhibited by the system at this wavelength, implying that the number of photons available for scattering by the growing polymer particles was the highest at 610 nm. Turbidity Measurements. Figure 1a presents changes in absorbance (for λ = 610 nm) of the reacting polymerization mixture during the 3 h interval over which the reaction was allowed to proceed. The two readings presented correspond to different amounts of n-butyl acrylate used in the initial reaction mixture expressed as volume ratios of the monomer to the total volume of the aqueous phase. The most conspicuous feature of the turbidity readings is the existence of different regimes in the recorded absorbance changes. During the first 250 to 500 s, the oxygen initially present in the system is removed by interactions with the PEGbased radicals. The turbidity then increases as the monomersaturated, SDS-based micelles begin to undergo polymerization, thus forming the first polymer particles. The prominent dips in the readings most probably correspond to instants in which the original emulsified monomer droplets become depleted, therefore leaving only the monomer absorbed on the latex particles as a material source for subsequent polymerization. Because the monomer concentration in these reservoirs is relatively high, the polymerization rate increases considerably after the dips and the absorbance rapidly reaches the value that characterizes the final emulsion. Consequently, toward the end of the reaction there are no more pronounced changes in absorbance. In general, as the ratio R increases, so does the turbidity of the latex emulsion at the end of the reaction, as shown in Figure 1b. Additional information about the properties of the latexes can be obtained from the DLS measurements. Stable latexes with particle diameters of between 100 and 3000 nm were obtained, as shown in Figure 1c. Monomer Conversion. The masses of the dry final products (Figure 1d) together with the results of the NMR measurements (Supporting Information) were used to estimate the monomer conversion, the degree to which the polymers were hydrophilic and the degree of polymerization. The results compiled in Table 1 point to the expected decrease in the

hydrophilicity of the product with increasing amount of monomer in the reaction mixture. In addition, the 1H NMR spectra (SI, Figures S1 and S2) show that the purified polymer is relatively free of monomer impurities because they exhibit no peaks in the vinyl proton region (5−7 ppm). Vesicle Formation. Microscope observations of the product prepared from the mixture characterized by ratios 0.029 through 0.057 revealed the presence of numerous vesicles in the water phase (Figure 2); their sizes did not exceed 10 μm, with the majority possessing diameters of between 3 and 6 μm (Figure 3). The other mixtures yielded products that did not produce any vesicles when subjected to the same protocol, which might be an indication of their hydrophobicity being too high. The size distribution of the vesicles was well approximated by a Frechet distribution with the following parameters: shape parameter α = 0.2221 ± 0.0952, scale parameter s = 1.0886 ± 0.1067, location parameter m = 2.8375 ± 0.1317, and χ2 = 9.4446. The mean value of the diameter was 3.75 μm with a standard deviation of 1.84. The dependence of the type of structure that a given copolymer can form on its physicochemical properties originates, as was mentioned above, in the hydrophobic-tohydrophilic balance of the amphiphilic molecule, more specifically the packing parameter, which depends on the relative sizes of the two parts of the molecule.8,27 As an example, for the copolymer poly(ethylene glycol)-block-poly(1,3-butadiene) when the ratio of the mass of the hydrophilic part of the chain to the mass of the entire molecule is between around 0.3 to 0.5, the expected outcome in terms of the selfassembled structures is going to be bilayers, which are potentially able to close and form vesicles.7 For larger values of the ratio, the products are too hydrophobic, and, ultimately, phase separation is observed. An analogous dependence is observed here for the situation where poly(n-butyl acrylate) acts as the hydrophobic block (the fourth column in Table 1). (It is interesting that in this context the glass-transition temperature, Tg, of poly(n-butyl acrylate) is relatively low and very close to that of poly(1,3-butadiene): −54 and −58 °C, respectively.) In the literature, vesicles of diameters in the range between 1 and 100 μm are called giant vesicles. From a purely taxonomical perspective, the vesicles obtained in the present study belong to this group, albeit at the lower range of diameters. However, because (a) the diameter range spans two orders of magnitude and (b) the inverted emulsion method has been successfully employed to produce polymersomes of diameters on the order of tens of micrometers,25 it would be interesting to understand why the vesicles reported here are in the lower range of sizes. It should be noted that in the present work we did not have in mind any specific target figure for the size of the resulting vesicles. Instead, we aimed here to demonstrate the general feasibility of obtaining vesicle-forming polymers with our experimental protocol. Furthermore, the sizes of the vesicles accessible with the inverted emulsion method depend on several factors that include the nature of the organic solvent, the size of the droplets present in the starting water-in-oil emulsion, the centrifugation time, and the relative centrifugal force (RCF).28 Because for the RCF and centrifugation times that we used (2.25 kG and 10 min, respectively) the size distribution of the vesicles is not expected to be very different from that of the emulsion droplets (Figure 3), we assume that the vigorous shaking and vortex mixing employed during the emulsion formation phase (that leads to the fragmentation of the larger

Table 1. Product Properties for Different Starting Amounts of n-Butyl Acrylate V0ButA (mL)

ratio R

mprod (g)

ratio Q

DP

conversion (%)

0.1 0.2 0.4 0.6

0.029 0.057 0.115 0.172

0.158 0.224 0.386 0.634

0.358 0.337 0.170 0.120

21 53 57 86

94 52 64 54 11269

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Figure 2. Sample optical microscope snapshots with polymer vesicles formed by the emulsion transfer method from the polymerization mixture characterized by R = 0.057 (a, b) and 0.029 (c). The scale bars are all 50 μm.

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CONCLUSIONS AND OUTLOOK Motivated by their self-assembly properties, we have described a new, simple method for synthesizing AB amphiphilic block copolymers. The method does not require tight environmental controls and uses a straightforward redox-initiated polymerization protocol that can be easily implemented. The properties of the products can be tuned prior to the synthesis by an appropriate selection of the starting acrylate concentrations. A standard emulsion polymerization protocol conducted under air at 40 °C yielded a product that, when the volume ratio of monomer to the aqueous phase was relatively low, was indeed shown to form vesicles. For the chosen conditions, the vesicle size was between 1 and 10 μm with an enhanced population at 3 μm which is well fit by the Frechet distribution. This is also the first report of the self-assembly of PEG-PnBA into giant vesicles. Finally, the work reported here gives access to polymersomes from inexpensive starting materials and may be of interest for studies of low-pH chemistry entrapped in polymersomes.30

Figure 3. Distribution of vesicle sizes for the polymerization mixture (in blue) characterized by R = 0.057 subjected to the emulsion-transfer method. The total number of vesicles used in constructing this distribution was 350. The distribution was well-described by the Frechet distribution (in red). The mean value of the diameter was 3.75 μm with a standard deviation of 1.84. The green dashed line represents the size distribution of the starting water-in-toluene emulsion droplets subjected to the inverted-emulsion vesicle formation protocol obtained from the DLS measurements.

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ASSOCIATED CONTENT

S Supporting Information *

Materials, a detailed description of the vesicle formation procedure, and a detailed description of the methods used in estimating the properties of the reaction products. This material is available free of charge via the Internet at http://pubs.acs.org.

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droplets) is the primary factor responsible for the bias observed in vesicle sizes. Vesicles formed from poly(ethylene glycol)-block-poly(nbutyl acrylate) were reported in the literature previously,29 but their sizes were considerably smaller than that reported in the present work (on the order of 100−1000 nm). To the best of our knowledge, our contribution is the first report of the formation of giant vesicles from this particular amphiphilic copolymer. We expect that the protocol described here can be generalized to other vinyl monomers, such as styrene and methacrylates. However, it is anticipated that the vesicle formation process in these instances will be affected by the much higher glass-transition temperature exhibited by their respective polymers.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Tereza Pereira de Souza for helpful discussions and suggestions and Dr. Jorge Carballido-Landeira for help with instrumentation. This work was funded by Repsol, S.A. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The DLS measurements were performed at the Center for 11270

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Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN) that is supported by the National Science Foundation under NSF award no. ECS-0335765. CNS is part of Harvard University.

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