Techniques To Control Polymersome Size - Macromolecules (ACS

Perspective pubs.acs.org/Macromolecules

Techniques To Control Polymersome Size Regina Bleul,† Raphael Thiermann,† and Michael Maskos*,†,‡ †

Department Nanoparticle Technologies, Fraunhofer ICT-IMM, Carl-Zeiss-Str. 18-20, 55129 Mainz, Germany Institut für Physikalische Chemie, Johannes Gutenberg-Universität Mainz, Jakob-Welder-Weg 11, 55128 Mainz, Germany

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ABSTRACT: Polymersomes as synthetic analogues of liposomes appear frequently in relevant literature as promising candidates for a wide range of different applications including drug delivery, theranostic multitools, and nanoreactors. In particular, as nanotransporters for nanomedical applications in vivo, requirements concerning the reproducible manufacturing and reliable size control are extremely high. This Perspective highlights the importance of size control especially in the context of nanomedicine and gives an overview of the theoretical background of amphiphilic self-assembly leading to different preparation methods, where their feasibility of controlling polymersomes’ size is discussed.

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INTRODUCTION Polymersomes are vesicular structures formed from block copolymers. Their naming has been adapted from the terms polymer and liposomes, their natural example built from phospholipids. The groups of Meijer in The Netherlands and Eisenberg in Canada broke the first ground reporting on polymeric vesicles for the first time in 1995.1,2 The direct comparison of polymersomes and liposomes concerning their membrane properties was presented 5 years later by Discher and co-workers.3 Polymersomes, compared to liposomes, provide enhanced variability as well as improved physical and chemical stability due to the good synthetic control in polymer chemistry.3−6 As liposomes, they enable dual loading with a hydrophilic inner lumen where sensitive, water-soluble agents can be encapsulated as well as a hydrophobic part of the polymersomes’ membrane where lipophilic cargo can be embedded.7−9 Different types of cargo as drug molecules or dyes and even inorganic nanoparticle as iron oxide or gold colloids have been combined within polymersome structures, leading to hybrid systems for versatile nanomedical applications.10−13 Triggering the membrane properties e.g. by external or internal stimuli enables a controlled release of a therapeutic cargo.14 Moreover, incorporation of tunnel proteins in polymersome membranes leads to smart nanoreactors with active transport through their membrane.15,16 Surface functionalization of polymersomes with specific targeting ligands additionally helps to transport e.g. drug-loaded vesicles to the diseased tissue.17−19 Typical preparation methods of polymersomes from amphiphilic di- or triblock copolymers include film rehydration and solvent displacement techniques.20,21 Most methods still suffer from low reproducibility and the feasibility for upscaling, which stays a big issue with regard to the translation into clinical application. The control over vesicle size plays a crucial role developing polymersomes for nanomedical applications. Polymersomes can © XXXX American Chemical Society

act as therapeutic tools carrying drugs or genes or as diagnostic probes enabling multimodal imaging.13,22,23 Entering the body they interact with a physiological environment including proteins and cells. In the context of cancer treatment the so-called enhanced permeability and retention effect (EPR effect), first described by Maeda et al.,24 is frequently reported. According to the EPR effect tumor vessels differ from healthy blood vessels concerning their architecture, namely endothelial cells in tumor tissue display wide fenestrations, so nanotransporters of a certain size can penetrate the blood vessel wall (Figure 1). Thus, nanotransporters accumulate selectively into tumor tissue (also called passive targeting) while small molecules diffuse unselectively into tumor as well as healthy tissue. Not only in tumor tissue but also e.g. in inflammatory regions an enhanced permeability of the blood vessel wall was observed; therefore, the application of polymersomes as nanotransporters is also for the treatment of infections very promising.25 Apart from the EPR effect, the size of polymersomes affects their fate in vivo in a variety of ways. Based on the assumption of intravenous injection as common practice in cancer therapy, nanotransporters will become in contact with serum proteins, and an opsonization will occur immediately. As in the literature reported, the interaction of serum proteins with nanoparticles depends besides the material properties and surface character also significantly on the size of the particles.26 The particle size as well as opsonization influences how fast the elimination of nanotransporters by the mononuclear phagocyte system will occur.27 Common practice for liposomes is to incorporate PEGylated phospholipids to reduce opsonization and prolong their blood circulation time. Polymersomes are often prepared Received: July 8, 2015 Revised: October 2, 2015

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DOI: 10.1021/acs.macromol.5b01500 Macromolecules XXXX, XXX, XXX−XXX

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Figure 1. Why size matters? The fate of nanotransporters inside the body is strongly influenced by the particles’ size (left). The EPR effect (enhanced permeability and retention effect) according to Maeda et al.24 (right). Nanotransporters (in contrast to small drugs) are not able to penetrate intact endothelium of healthy tissue. The enhanced permeability of tumor endothelium, due to larger fenestrae, allows an extravasation of the particles. Because of insufficient lymph drainage in tumor tissue, nanotransporters remain in the tumor tissue for a prolonged time (retention).

from block copolymers with PEG as hydrophilic block. Brinkhuis et al. reported on significant differences in blood circulation times of polymersomes, from the block copolymer polybutadiene-block-poly(ethylene glycol), with diameters between 90 and 250 nm.28 It was found that 30% of the small polymersomes with diameters of 90 nm circulated even after 24 h, whereas polymersomes above 120 nm were mostly cleared from the bloodstream after 4 h. Besides the phagocytic cells also the vital organs decide size selectively on nanocarriers’ fate.29 For example, it has been shown that liposomes of more than 200 nm accumulate in the spleen and liver, whereas liposomes of less than 70 nm were predominately found in the liver.30,31 This size selectivity of the organs can to some extent be attributed to their specific dimensions. For example, the lung capillaries are the smallest blood vessels inside the body with diameters of about 2−13 μm;32 big particles or aggregates of smaller particles can get stuck there; particles smaller than 4−6 nm can be excreted by the kidney;32 the endothelial fenestrations of the liver are in the region of 100−150 nm.33 The blood−brain barrier is a highly protective barrier formed by brain endothelial cells connected by tight junctions, which is generally not negotiable for nanoparticles. Nevertheless, specific surface modifications have been shown to be promising to help particles smaller than 60−80 nm to overcome this barrier.34−36 Taking into account that size is a crucial factor concerning the fate of nanotransporters inside the body, the development of appropriate preparation methods for polymersomes as nanopharmaceuticals is of highest importance. Reproducibility and cost-efficient large-scale manufacturing are basic requirements for the transfer into clinic.

morphologies like spherical and cylindrical micelles or vesicular structures are formed preferably.38−40 For small molecule amphiphiles Israelachvilli et al. published in 1976 their theory of self-assembly and introduced the concept of critical packing parameter (Pc) to predict the assembled morphologies.41 The critical packing parameter describes the geometry of an amphiphile and is defined as Pc = v/a0lc, where v is the volume of the hydrophobic chain, a0 is the area occupied by the hydrophilic headgroup, and lc is the length of the molecule (Figure 2).

THEORETICAL BACKGROUND OF BLOCK COPOLYMER SELF-ASSEMBLY Amphiphilic block copolymers are able to microphase separate and form self-assembled nanoscale structures in bulk (melt) and in solution.37 Depending on the ratio between the hydrophilic and hydrophobic part of the polymer, different

Amphiphiles with Pc values below 0.5 result in highly curved aggregates such as spherical and worm-like micelles, while with increasing Pc values until v/a0lc = 1 bilayer formation is favored. This geometrical consideration of amphiphiles can to some extent be translated to the self-assembly behavior of block copolymers. Here the hydrophilic fraction ( f hydrophilic) is commonly used to predict the expected morphology. Polymer-

Figure 2. Critical packing parameter Pc and the resulting morphologies of amphiphile aggregates according to Israelachvili et al.41

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Figure 3. Two mechanisms of vesicle formation from initial homogeneous state. Mechanism 1: small, spherical micelles are formed. They grow by collision to larger anisotropic (cylindrical or disk-like) micelles. Finally, disks close up to vesicles. Mechanism 2: similar to mechanism 1. First spherical micelles are formed and then grow up to larger spherical micelles. Solvent diffuse inside those micelles, and vesicles are formed. Scheme adapted from ref 46.

vesicles. Finally, rearrangements of solventphobic and solventphilic copolymer segments occur, while more solvent diffuse into the semivesicles and full vesicles form.46,47 It should be noted that the mechanism of vesicle formation influences the encapsulation efficiency. Therefore, according to mechanism 1, a statistical encapsulation of compounds from the surrounding solvent ought to be enclosed; compared to this formed vesicles as described in mechanism 2 would have a lower loading efficiency. There are evidencing experimental results for both pathways in the literature;48,49 thus, the proceeding mechanism is apparently strongly dependent on the polymer and solvent properties as well as the exact conditions. Kinetic of Vesicle Formation. Optimizing preparation strategies for polymersomes requires besides the knowledge of possible mechanisms also an understanding of the kinetics of the self-assembly process. Vesicle formation from the nanometer to micrometer scale proceeds during milliseconds or even microseconds depending on the molecular weight of the amphiphiles. Intermediates can therefore be hardly experimentally observed. High performance simulation tools as molecular dynamics (MD) enables nowadays to simulate the initial states of vesicular self-assembly. The formation of small surfactant micelles and small vesicles with diameters of 10−20 nm has already been studied with atomistic MD.50,51 MD simulations on fully atomic models are limited to small membrane sections of a few ten nanometers because otherwise the time scale for the self-assembly process in particular for large amphiphile systems is too long and cannot be studied with currently available computers. In a recent publication of Shillcock et al. simulations using parallel dissipative particle dynamics (DPD) following the spontaneous vesicle formation with near-molecular resolution were presented.52 An amphiphile with linear architecture H3T6, in which three hydrophilic “head” particles (H) are attached to a chain of six hydrophobic “tail” particles (T), was chosen as a representative model molecule. This molecular architecture had already been shown to form stable bilayer structures.53

somes are observed at hydrophilic weight fraction 100). For the description of microphase separation in the presence of a selective solvent further Flory−Huggins interaction parameters has to be considered which includes the interaction between the A and the B segment and between each segment with the solvent. The self-assembly process of amphiphiles in the presence of an additional cosolvent is even more complex. Mechanisms of Vesicle Formation. Regarding the process of vesicle formation in solution, two different mechanisms are currently discussed (Figure 3).46 Both postulated a mechanism starting from amphiphiles as unimers. Then small micelles or polymer clusters are formed. Mechanism 1 describes the growth of these micelles to sheetlike bilayers, which tend to closure into vesicular structures driven by the line tension.43,46 In contrast to mechanism 1, mechanism 2 describes the growth of the micelles to bigger spherical micelles with a solventphilic core, so-called semiC


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Figure 4. Time series of snapshots of vesicle self-assembly from an initially random dispersion of 58 240 amphiphiles in 5 000 000 water particles contained in a (90 nm)3 simulation box (80 mM amphiphile concentration). Amphiphiles have the linear architecture H3T6; the hydrophilic H particles are colored red and the hydrophobic tail particles orange, except that the terminal tail particle is green. These snapshots illustrate the three stages of growth. Initially, the amphiphiles form small micelles that diffuse, merge, and transform into planar bilayer patches (a, log(time) = 9.9, ∼2 μs). Next, a middle phase of growth appears during which the bilayer patches grow and curl up into vesicles that subsequently continue to grow by fusion with each other and the remaining bilayer sheets (b, log(time) = 13.6, ∼80 μs). Eventually the system is dominated by closed vesicles that diffuse around but only occasionally fuse as the vesicle fusion process requires times longer than the current simulations (c, log(time) = 14.3, ∼162 μs). Reproduced with permission from ref 52.

water. The detergent is subsequently slowly removed by dialysis.61 Polymersome preparation by the cosolvent method or also called solvent displacement or nanoprecipitation method begins with the amphiphilic polymer dissolved in a water-miscible solvent. The polymer solution is added dropwise into water under vigorous stirring.62 The solvent is subsequently removed by dialysis, freeze-drying, or evaporation. The inverse technique with the successive addition of water into the polymer solution until structure formation is induced can also be performed.8,63 Conventional methods such as film rehydration or direct dissolution of bulk material in general produce a wide range of vesicle sizes. Thus, often postpreparation treatments like extrusion through a polycarbonate membrane with defined pores, sonication, or freeze−thaw cycles are required to obtain an appropriate size distribution of vesicles.20,64,65

Snapshots of a parallel DPD simulation of vesicle selfassembly from an initially random dispersion of model amphiphiles in water are shown in Figure 4. The process of vesicle self-assembly can be described in three successive stages, corresponding to mechanism 1 earlier mentioned. The stages are dominated by different aggregates that grow by distinct kinetic mechanisms. Initially, small micelles are formed very rapidly. Earlier publications based on MD simulations revealed a few nanoseconds for this process.54 During the second stage the micelles grow and transfer into quasi-planar bilayers. Next, micelles and smaller bilayer fractions diffuse and merge to larger sheets that finally closure into vesicular structures. The duration of this stage is longer since the diffusion velocity of aggregates is slower than diffusion of unimers. For small amphiphiles this phase continues about 50−400 μs depending on the concentration. The fusion of large vesicles proceeds on an even longer time scale and therefore exceeds the simulation time. Concerning a potential loading of the vesicular structures, the second stage, namely the growth of the bilayer sheets, appears to be the crucial stage.55

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SIZE CONTROL The above-mentioned cosolvent method or solvent displacement method is a commonly used technique for polymersome preparation and enables to influence the polymersome size and morphology in a variety of ways. Important pioneer work and many experiments around block copolymer self-assembly arise from the group of Eisenberg.2,66,67 In solution, the self-assembly of thermodynamically stable block copolymer aggregates is an interplay of different forces: (i) the degree of stretching of the core-forming blocks, (ii) the interfacial tension between hydrophobic blocks and the solvent outside the core, and (iii) the repulsive interactions among corona forming chains.67 In the following sections, the factors affecting the selfassembly of block copolymers are briefly presented. The parameters to control size and morphology of block copolymer aggregates include the copolymer composition, the initial copolymer concentration, the nature of the common solvent, the water content in the solvent mixture, the temperature, and possibly the addition of other components as ions, homopolymers, or surfactants.68 A typical cosolvent experiment starts with a copolymer solution in a suitable solvent favorable for both blocks. Successively addition of water, as precipitant for the hydrophobic block, induces the self-assembly at a critical water content. The system can be quenched by the sudden addition of a large excess of water, which leads to strongly limited chain mobility and a freezing of the current morphology.69 This applies in particular to polymer systems with the hydrophobic

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POLYMERSOME PREPARATION METHODS Most conventional batch methods commonly used for polymersome preparation are adapted from liposome preparation techniques. The film rehydration is a frequently applied method for lab-scale production of liposomes or polymersomes. Starting from a block copolymer solution in an organic solvent, a thin polymer film on a round-bottomed flask is formed by evaporation. The rehydration of the polymer is achieved by subsequently adding an aqueous buffer, which leads to a detachment of the film from the glass surface. The swelling process can be influenced by stirring, shaking, or sonication, which affects to some extent the resulting vesicle size. In general, this method results in unilamellar as well as multilamellar vesicles with a rather broad size distribution.56−58 Some amphiphiles also allow a direct dissolution from bulk material; however, a longer and much more vigorous agitation is required to obtain the complete rehydration of the polymer.20,59 Electroformation is a derived method of film rehydration. The polymer is hereby spread on a pair of electrodes, and the rehydration process in aqueous buffer is induced under an oscillating electric field. Rather uniform giant unilamellar vesicles (GUV) in the micrometer range are commonly obtained. 3,20,60 The detergent method uses surfactants to assist the direct dispersion of the polymer in D


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segments, which is connected with the packing parameter similar to small amphiphiles.68 Yu and Eisenberg demonstrated the formation of different aggregate morphologies and dimensions from a PS-b-PAA block copolymer system with N,N-dimethylformamide, tetrahydrofuran, and dioxane as well as mixtures for those as initial solvent.66,67,78 In this case, with lower polarity of the solvent, the weaker the PAA−solvent interaction and the weaker the repulsive interactions among the corona chains, thus the aggregation number and the degree of stretching increase. This can be summarized as follows: The more similar the solubility parameter of the solvent and that of the core forming block, the higher the solvent content of the core and the higher the degree of stretching of the core chains. Regarding the polymersome preparation by the cosolvent method, the influence of the common solvent is almost negligible, if the polymer solution is injected in the water phase, which is in a large excess.77 Obtained vesicles exhibit a rather broad size distribution due to the rapid self-assembly forced by the unfavored interaction of hydrophobic chains and water. Performing the opposite order of addition, the nature of the solvent as well as the duration of addition influences the vesicle size. Hereby diffusion plays a major role, which e.g. depends on solvent−water miscibility, viscosity, and polymer solubility. In the case of homopolymer nanoprecipitation Galindo et al. demonstrated a correlation between the solvent−water Flory− Huggins interaction parameter (χsolvent−water) and the average size of the obtained nanoparticles.79 Sanson et al. presented their results of polymersome preparation from poly(trimethylene carbonate)-b-poly(L-glutamic acid) initially dissolved either in DMSO or in a THF/methanol mixture.77 Vesicles obtained from a THF/MeOH polymer solution exhibit with an average hydrodynamic radius of about 200 nm double the size compared to the polymersomes starting with polymers dissolved in DMSO (rh = 100).77 According to the authors, the solvents having a high affinity to water (low χsolvent−water values) facilitate a fast solvent diffusion into the water phase, leading to a rapid quenching of polymer aggregate growth (χsolvent−DMSO = 16.35 < χsolvent−THF/MeOH = 17.45). The influence of the water content can be described in a similar manner. As the water acts as a precipitant for the hydrophobic block at a critical water concentration, unimeric polymer chains start aggregating. The critical water content depends on the molecular weight, the polymer concentration, and the nature of the common solvent.67 With an increasing amount of water, the morphologies of the aggregates tend to transform from spheres via rods or disks to vesicles, driven by the interfacial energy contribution to the free energy.76 With increasing water content the system minimizes the total interfacial area by increasing the vesicle size. The speed of water addition determines how slow or fast the solvent quality changes and the duration unimers can diffuse. Thus, low flow rates lead to higher aggregation numbers and larger vesicles.77 Influence of Additives. The presence of additives as ions, homopolymers, and surfactants can also provide control over vesicle formation. Zhang and Eisenberg demonstrated the transition from spheres to rods into vesicles, adding an increasing amount of CaCl2, HCl, or NaCl for example to a PS410-b-PAA25 copolymer/DMF solution.80 This transition can be explained by changes of the repulsive interactions among the hydrophilic PAA chains. Burke and Eisenberg have shown that with the addition of a surfactant, sodium dodecyl sulfate (SDS), to the PS310-b-PAA53 copolymer solution the required water

blocks having a high glass transition temperature (Tg), as for example PS-b-PAA.70 Basically, the thermodynamic equilibrium is only reachable, when the preparation conditions as the content of common solvent and the temperature enable a sufficiently high mobility of the polymer chains and morphologic transitions can occur faster than the addition of water. In many cases thermodynamic equilibrium is unreachable over the experimental time scale; thus, aggregate formation occurs under kinetic control and is strongly dependent on the mechanism of aggregate formation.70 Size Control on Polymer Level: Influence of the Polymer Composition. The size of polymersomes can be influenced at different stages of their preparation. For instance, Wang et al. demonstrated how the size of unilamellar polymeric vesicles from palmitoyl glycol chitosan was controlled by polymers of different molecular weights.71 Vesicles with zaverage mean diameters in the range of 200−480 nm were obtained after sonication of polymers with molecular weights between MW = 31 000 and 276 000, whereby low molecular weight polymers resulted in smaller vesicles. Terreau et al. reported the effect of polydispersity on vesicles size.72 PS-bPAA copolymers with different PAA block polydispersity were investigated concerning their aggregate morphology. Generally, a decrease of vesicle size with increasing PAA polydispersity index was observed. Luo and Eisenberg explained this effect with a segregation of the long chains preferentially to the outside of the vesicle while the short chains segregated to the inside of the vesicle.63 A further influencing factor of vesicle size is the block copolymer composition, which affects not only the morphology but also the polymersome size to some extent.66 Greenall and Marques demonstrated how the curvature of polymersome membranes and with that the vesicle radius can be controlled by the copolymer architecture.73 They designed tetrablock copolymers with the aid of coarse-grained calculations that formed vesicles of a predetermined size. Influence of the Initial Copolymer Concentration. The effect of variation the initial copolymer concentration was among others shown using the example of PS410-b-PAA24 in DMF.74 With increasing polymer concentration from 2 wt % spherical aggregates were found, at 2.6% cylindrical micelles were formed, and at 4% predominately vesicles with sizes ranging from 50 to 500 nm were present. The increase of copolymer concentration also results in an increased aggregation number. The importance of polymer concentration for the vesicle forming was also shown in phase diagrams of different PS-b-PAA.75 For a PS310-b-PAA52 block copolymer system at a water content of 25 wt % vesicle formation only occur at polymer concentrations above 0.6 wt %.76 With increasing polymer concentration from 0.6 to 5 wt % vesicle sizes increased from 90 to 124 nm. The same effect was demonstrated with poly(trimethylene carbonate)-b-poly(Lglutamic acid) polymersomes starting from copolymer concentrations in DMSO from 1 to 100 mg/mL, resulting in hydrodynamic radii between 72 and 176 nm.77 It should also be mentioned that the increase in vesicle size with an increasing initial amphiphile concentration was also observed in the DPD simulation by Shillcock.52 Influence of the Solvent Nature and Water Content. The common solvent dissolving both the hydrophobic and hydrophilic blocks also influences the morphology and size of the aggregates.66,67,77,78 The solvent−polymer interaction determines the relative coil dimensions of the both polymer E


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Figure 5. Images of the polymer islands, the vesicles formation process, and vesicle size distributions. (a) 3D image (generated from a series of vertical slices) of the vesicle-forming surface showing the swollen exterior bilayer before detachment. (b) A single vertical slices showing a series of vesicles “budding” form the surface. (c) Differential interference contrast optical microscopy of the dewetted surfaces following spin-coating using 2000 mesh (i) and 1000 mesh (ii) TEM grids. (d) Mass-normalized frequency of vesicle size distribution for the patterned surfaces shown in (c). Reproduced with permission from ref 86. Copyright 2009 Macmillan Publishers Ltd.

yielded unilamellar polymeric vesicles with distinct sizes depending on the utilized template (Figure 5). A further development of the cosolvent method was recently reported by Förster and co-workers.87 The authors describe the use of inkjet technology of printers to prepare small unilamellar vesicles below 200 nm in size by injecting picoliter volumes of polymer solution under stirring in water. This technique results in vesicles with a relatively narrow size distribution and has been shown to be well reproducible. However, this technique is limited to polymers soluble in ethanol due to the lack of chemical resistance of the cartridges for other solvents. A very recent publication by Hickey et al. reported on size control of polymersomes by the incorporation of different sized magnetic nanoparticles.88 The self-assembly of a micelleforming polymer PAA38-b-PS73 was performed in the presence of hydrophobic iron oxide nanoparticles with different sizes. As already previously reported, the incorporation of hydrophobic nanoparticles can induce a partial transition of the morphology from micelles to vesicle.89 In the case of PAA38-b-PS73 the incorporation of iron oxide particles results in a mixture of hybrid micelles and polymersomes. Interestingly, varying the size of incorporated nanoparticles effectively influenced the yield of polymersomes as well as their size. Hereby also the localization of the nanoparticles was size-dependent; small nanoparticles were dispersed in a polymer bilayer, and large particles were found at the interface between the inner and outer layer of a bilayer membrane. The authors hypothesized

content to induce a morphology change of the aggregates was lowered.81 This effect is based on two different interactions: the screening of the electrostatic charge of the PAA chains by the sodium ions and the interaction of the hydrocarbon tails with the hydrophobic aggregate core. The effect of the addition of homopolymer was also investigated using different PS-b-PAA copolymers. The addition of homopolystyrene to the diblocks changed the morphologies from bilayers or cylinders to spheres, while spherical micelles did not change their morphology but increased in size, due to accumulation of homopolymer in the hydrophobic core.82 Sui et al. reported on the addition of oligoethylene glycol to PEG−polycaprolactone (PCL) block copolymers to achieve a transition of spherical micelles to polymersomes.83 The polymersome preparation was performed via direct dissolution at high temperature without an additional solvent but with an excess of oligo-ethylene glycol. Size Control during Vesicle Formation Process. Several methods to obtain size-controlled polymersomes have already been reported; nevertheless, they are often limited to vesicle sizes the micrometer range.84,85 Howse et al. presented an interesting approach of template formation of micrometer-sized vesicles by photolithography.86 Both 1000 and 2000 mesh transmission electron microscopy (TEM) grids were used to generate a patterned hydrophilic/ fluorocarbon surface. The polymer solution was spin-coated, resulting in polymer islands. The rehydration from these grids F


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Figure 6. Left: (a−c) TEM images of magneto-polymersomes assembled with (a) 6.4, (b) 10.8, and (c) 16.3 nm iron oxide particles at 25 np wt %. Blue and red arrows indicate polymersomes and micelles, respectively. (d) Size histogram of magnetopolymersomes determined by TEM. (e) Pictorial description showing how the nanoparticle size affects the polymersome population and dimension. Right: reconstructed 3-D surface rendering of the tomographic volume and X−Y computational slices (0.23 nm) at the midpoint of magneto-polymersomes assembled with (f) 5.8, (g) 9.9, and (h) 16.3 nm iron oxide particles. Scale bars are 100 nm. (i) Pictorial representation showing how the nanoparticle size affects the curvature of polymersome membranes. Reproduced with permission from ref 88.

they are too big for most drug delivery applications, particularly for systemic administration by intravenous injection. Microfluidic Approaches. Recently different microfluidic setups for the continuous preparation of polymersomes have been reported.93−97 Most of these microchips are based on a simple Y-junction and run with small flow rates in the μL/min range. In 2005, the group of D. Weitz presented for the first time a coaxial flow microfluidic device for polymersome preparation that generates highly uniform, monodisperse giant polymersomes in the micrometer range by the water-in-oil-in-water double emulsion technique.98,99 A schematic drawing of this microfluidic device with its three different fluid phases is shown in Figure 7.99 The middle phase consists of the diblock

the size-dependent effect on the self-assembly is to some extent an entropic effect of inserting large nanoparticles into the polymer domain and additionally a geometrical effect of the asymmetric layer structure of the vesicle membranes resulting from a size-dependent nanoparticles distribution in the membrane (Figure 6). As earlier discussed, also the copolymer composition can influence the vesicle size, which is related to the vesicle curvature that is stabilized by preferential segregation of hydrophilic blocks by length.72,82 For the herepresented example this implies due to the selective localization of different sizes nanoparticles, the curvature of the membrane increases by the incorporation of larger particles leading to smaller polymersomes (Figure 6). In recent literature an increasing number of publications about so-called vesosomes appear in the context of nanomedicine. Vesosomes are a type of self-assembled multicompartment structure that is designed to protect and retain encapsulated drug molecules through two successive membranes. For instance, for ciprofloxacin it was demonstrated that a double lipid membrane of vesosomes increased the serum half-life of the drug by a factor of 36 compared to the single liposome.90 Vesosomes based on block copolymers can for example be prepared by combinations of different standard preparation methods such as film rehydration and solvent displacement but generally suffer from low encapsulation efficiencies.91 Marguet et al. reported on polymer vesosomes prepared by a combination of nanoprecipitation and emulsion centrifugation.92 An emulsion of aqueous sucrose polymersome dispersion in toluene stabilized by another block copolymer is added to an aqueous glucose solution. By centrifugation the droplets are forces to cross the interface covered with further block copolymers, and finally giant polymer vesosomes are formed. The diffusion barrier of the double membrane of these micrometer-sized vesosomes was evidenced with doxorubicin as a model drug encapsulated in single polymersomes and compared to doxorubicin-loaded vesosomes. Even though vesosomes enable an encapsulation of multiple and distinct ingredients and allow an enhanced control of the release profile,

Figure 7. Schematic of the microcapillary geometry for generating double emulsions. The geometry requires the outer phase to be immiscible with the middle phase, which is in turn immiscible with the inner phase. Both the injection tube and the collection tube are tapered from glass capillary tubes with an o.d. of 1000 μm and an i.d. of 580 μm. Typical inner diameters after tapering range from 10 to 50 μm for the injection tube and from 40 to 100 μm for the collection tube. Reproduced with permission from ref 99.

copolymer dissolved in an organic solvent. Water and the hydrophilic cargo (if any) are located in the middle phase. In this case a viscous aqueous solution of alginates represents the outer phase. The fluid streams of the inner and middle phase are hydrodynamically focused on the orifice of the collection tube to form uniform W/O/W double emulsion droplets. The w/o/w emulsion technique in microfluidic devices is nowadays also used to generate giant magnetic polymersomes G


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Figure 8. Schematic representation of the controlled block copolymer self-assembly in a micromixer. The polymer solution (in a common solvent) is diluted with a selective solvent (water) in a microstructured mixing device, with increasing concentration of selective solvent in the polymer phase the polymersome self-assembly is induced. Resulting sizes (and morphologies) of the polymer aggregates can be controlled by varying different parameters as polymer composition, end-group functionality, mixing ratio, and flow rate. Subsequent surface functionalization, e.g. attachment of specific targeting moieties, is feasible. Moreover, an in situ loading can be performed by adding the cargo (e.g., drug molecules) to the starting polymer solution or the selective solvent. Depending on the hydrophilicity of these molecules, they will be incorporated in the hydrophobic part of the membrane or encapsulated in the hydrophilic inner lumen of the vesicle. The insets show two different micromixers with their corresponding flow profiles.

reproducibility is limited in batch methods, which is another important advantage of the continuous micromixer method. The procedure of polymersome preparation in a micromixing device is fairly simple as shown in Figure 8. Similar to the cosolvent method, the amphiphilic polymer is dissolved in the common solvent for all blocks. Cargo can be added into the initial polymer solution or into the water phase depending on its solubility and stability. The controlled addition of the selective solvent during the micromixing process induces the self-assembly. The obtained block copolymer aggregates are subsequently purified by dialysis, gel filtration, or simply by evaporation depending on the utilized polymer, solvent, and cargo. Additional surface functionalization, e.g., attachment of specific ligands for cancer cell targeting, broadens the range of potential applications in nanomedicine. Vesicle Formation in Micromixers: Different Phases of Self-Assembly. The self-assembly in a micromixing device can be pictured similar to the simulation results of Shillcock et al. (Figure 4). First block copolymers are present as unimers dissolved in the common solvent. The fluids of polymer solution and the selective solvent (water) join up in the micromixer. Initially polymer chains can diffuse freely until the critical water content is reached and aggregate formation begins. Depending on the polymer at a certain water content solubility of the unimers is so low that almost no diffusion is feasible and no exchange between the formed aggregates can occur. However, unimers of small surfactant molecules or polymers with a relatively high water solubility can diffuse freely even in pure water; thus, in this case the dynamic process of

as well as multicompartment polymersomes as the abovementioned vesosomes.84,100,101

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SIZE-CONTROLLED CONTINUOUS SELF-ASSEMBLY OF POLYMERSOMES IN MICROMIXERS Polymersome preparation in micromixers is as the abovementioned inkjet technology a further development of the cosolvent method. The concept of block copolymer selfassembly in a micromixing device was first described by Mueller et al. in 2009 using the example of poly(butadiene)-bpoly(ethylene oxide) (PB-b-PEO) diblock copolymers.102 Performing vesicle preparation in a micromixing device gains several advantages over the cosolvent batch method as well as the inkjet technology. While the vesicle size with the inkjet method is strongly dependent on the injected volume, the control over aggregate size and morphology in the micromixer can be controlled varying different parameters as e.g. the initial polymer concentration, the nature, the water content of the common solvent, the mixing ratios, and the total flow rates.102,103 Compared to the batch cosolvent method, where mixing efficiency is dependent on the batch volume as well as stirring velocity, micromixer technology enables a very efficient mixing. Thus, even self-assembly of polymers with a small hydrophilic fraction can be performed. Furthermore, the incorporation of strongly hydrophobic components that tend to precipitate in direct contact with water in the vesicle membrane is feasible.104 In general, throughput as well as H


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Figure 9. Phases of amphiphile self-assembly process in a micromixer depending on the flow rates. The transition from laminar to turbulent flow is shown by different patterned background.

Figure 10. TEM images of PB130PEO66 polymer vesicles and micelles: Self-assembly was performed with a caterpillar micromixer (model CPMMR300, Fraunhofer ICT-IMM) with copolymer solution in tetrahydrofuran/H2O (50/50% v/v) and symmetrical flow rates. Vesicles were obtained with a total flow rate of 1.2 mL/min (initial polymer concentration 3 g/L) (left) and predominately spherical micelles were obtained with 9.6 mL/ min total flow rate. Samples were γ-irradiated prior to TEM measurement to fix the polymer aggregates by cross-linking.

lower water contentthereby prolonging the time the micelles grow to disk-like double layers. Accordingly, the resulting vesicles exhibit a larger diameter than those starting at lower polymer concentration.52,103 Beside the factors as polymer composition, temperature and common solvent, that are dependent on the nature of the polymer and are also valid for the batch process, polymersome size can also be influenced by the mixing parameters as for example the flow rate. In general, micromixers based on multilamination mixing principles exhibit a laminar flow profile close to the inlet area. Under laminar flow conditions mixing time is based on

aggregate formation and transformation is often observed further on. Self-assembly in water is mainly driven by the so-called hydrophobic effect.105,106 The critical water content that determines the beginning of amphiphile aggregation depends on several factors: (i) polymer concentration, (ii) temperature, (iii) copolymer composition (monomers, block length and ratio, molecular weight), and (iv) the properties of the common solvent. The influence of the polymer concentration does not differ from the batch method. Thus, with increasing polymer concentration the aggregate formation starts already at a I


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Figure 11. Flow rate dependency of vesicle sizes. (A) Dynamic light scatting results of polymer vesicles (PB130−PEO66) depending on the flow rate for different mixing geometries: caterpillar micromixer CPMM (squares), superfocus interdigital SFIMM (circles), and slit interdigital micromixer SIMM (triangles). Reproduced with permission from ref 103. Copyright 2012 Elsevier Ltd. (B) Dynamic light scatting results of Pluronic polymersomes samples indicating a good control over vesicle size: Pluronic L121 was dissolved in THF (10 g/L) and mixed with water in mixing ratio 1:10 in a caterpillar micromixer. Hydrodynamic size can be controlled varying the total flow rate. Error bars showing standard deviation of mean value from five independent measurements. The dashed line is a guide to the eye.

erably forms lamellar aggregate structures.111,112 Because of its hydrophobicity, the polymer tends to precipitate rapidly in contact with water; thus, the preparation with the conventional cosolvent batch method is not feasible. Foster et al. reported the preparation of giant Pluronic vesicles with a double emulsion method.113 Vesicles smaller 200 nm have previously only been accessible by sonication in combination with extrusion.112,114 The continuous preparation of Pluronic with micromixers was recently reported.104 It was shown that polymersome with diameter below 200 nm were obtained, and even the challenging incorporation of strongly hydrophobic iron oxide nanoparticles was successful. In contrast to the above-mentioned PB−PEO polymer system, for which the most uniform vesicles were obtained with symmetrical flow rates starting with polymer in a THF/H2O premixture, Pluronic vesicles are only accessible starting with a polymer solution in pure organic solvent with asymmetrical flow rates. In Figure 11B our latest results concerning the flow rate dependency of vesicle sizes from Pluronic L121 are shown. We were able to adjust the mean hydrodynamic vesicle size from around 100 to 250 nm.

diffusion and thus dependent on the lamella thickness. Nevertheless, under realistic conditions internal friction often leads to turbulences that change the flow profile.107 The mixing speed in the turbulent region of the mixer is strongly increased compared to the laminar region. The transition from laminar to turbulent flow profile depends on the flow rate. Since the content of the selective solvent in the polymer phase is directly related to the mixing speed, vesicle formation can be effectively controlled by variation of the flow rate. A model showing the influence of the flow rate on the selfassembly process in the micromixer is presented in Figure 9. With increasing flow rate the transition from laminar to turbulent flow is shifted closer to the mixer inlet; thus, the structure formation is terminated at an earlier stage. In this way, even thermodynamically unfavored aggregate structures can be obtained. For instance, for a preferable vesicle-forming polymer prestage aggregates as spherical micelles or even disk-shaped bilayers can be kinetically trapped. Figure 10 shows different aggregate structures formed by the identical polymer PB130− PEO66 prepared under different flow rate conditions. This result leads to the assumption that practicable mixing times with the aid of micromixer technology are of the order of the kinetics of vesicle formation from larger block copolymers as the PB130−PEO66 used in this example. Varying the flow rate in a smaller window also vesicle size can be controlled.103 Figure 11A shows the flow rate dependency of the hydrodynamic radius of obtained vesicles from PB130− PEO66. There is a critical flow rate where no further decrease in size is observed, since morphology transition to spherical micelles has already occurred. The transferability of the size control in micromixers was also confirmed with a different polymer system poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide). Regarding potential applications in nanomedicine this material was chosen because of its biocompatibility and applicability for pharmaceutical application that have already been frequently shown.108−110 Pluronic L121(EO5PO68EO5) is a relatively hydrophobic PEO−PPO−PEO triblock copolymer that pref-

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CONCLUSIONS AND OUTLOOK Polymersomes represent an exciting and rapidly developing area of multidisciplinary research. In particular, in the context of nanomedicine polymersomes as nanotransporters appear frequently in the current literature because of their advantages over liposomes and enormous variability. As mentioned at the beginning of this Perspective, the size of the nanocarriers plays a critical role in the success of drug delivery systems. Nanotransporters with very small sizes 200 nm are at risk of being caught immediately by cells of the mononuclear phagocyte system and end up in the liver and spleen. Modern nanomedicine also addresses questions concerning a successful and controlled crossing of biological barriers. Control over size and morphology of polymer aggregates might be one key to success. The self-assembly of amphiphilic block copolymers is a complex interplay of several J


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factors starting from polymer composition to solubility parameters of the solvents and also includes concentration and temperature effects. A comprehensive understanding of the geometrical interactions, the thermodynamics, and the kinetics of self-assembly is the basis to adjust or create manufacturing techniques that allow controlling the self-assembly process. Many conventional methods arise from the liposome preparation and have been successfully transferred to polymersome preparation. Nevertheless, many of them result in a rather broad size distribution and are not suitable for upscale. In particular, reproducible large-scale manufacturing of polymersomes remains challenging. With regard to the translation into clinical application this would be one of the basic prerequisites. Many of the existing polymersome preparation techniques as for example direct dissolution, electroformation, and template formation by photolithography are small-scale batch methods. Some of them, in particular the last-mentioned, provide a relatively good size control but generally in the micrometer range. Impressing progress concerning the encapsulation of different functional materials as drugs including biological macromolecules, dyes, and imaging probes has been made and multimodal and multifunctional systems as e.g. the vesosomes has been developed. The example of Hickey et al. also showed how incorporated nanoparticles not only enhance the application field but also enable a control of polymer aggregate size and morphology. The microfluidic approaches belong to the continuous techniques; nevertheless, they are often limited to a quite low throughput in the range of μL/min. In particular, the double emulsion method by Weitz et al. provides the manufacturing of uniform vesicles and even complex structures as magnetic polymersomes and multicompartment vesicles. The size control in this case is limited to the dimensions of the microchannel and therefore also limited to the micrometer scale. The here-presented approach using micromixer technology has already been shown to enable a continuous preparation of polymersomes also with sizes below 200 nm. Rapidly mixing even allows to some extent a kinetic control over the selfassembly. Thus, besides the control over vesicle size, it is also possible to obtain thermodynamically unfavored polymer aggregates for example spherical micelles or bilayer disks as precursors of polymeric vesicles. The manufacturing of polymersomes in a micromixer additionally provides the possibility to perform an in situ loading with hydrophilic as well as hydrophobic substrates in a one-step process. In this way even hybrid vesicles from polymers and inorganic particles can be obtained. Taking the example of Pluronic vesicles with incorporated magnetite nanoparticles the broad range of potential biomedical applications becomes visible; this includes e.g. magnetically guided transport or magnetic field induced drug release. Magnetic polymersomes, using the encapsulated iron oxide nanoparticles as contrast agent for magnetic resonance imaging, are even applicable as theranostic tools that are suitable systems for monitoring drug delivery. The continuous and reliable straightforward preparation method with micromixers enables the production of welldefined nanotransporters with controllable size and morphology that can be individually designed. Previous achievements let us be confident that in future also for large-scale production of nanopharmaceuticals micromixer-based plants take on greater significance in the pharmaceutical industry.

Perspective

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (M.M.). Notes

The authors declare no competing financial interest. Biographies

Regina Bleul studied biotechnology at the University of Applied Sciences of Darmstadt. In 2010 she joined the group of Prof. Maskos at the Federal Institute of Materials Research and Testing and worked on her PhD thesis on the development of polymersomes as multifunctional drug carriers. In 2012, she went to a research stay at the University of British Columbia, Vancouver, as DAAD fellow and joined there the group of Assoc. Prof. Urs O. Haefeli in the faculty of pharmaceutical sciences. After completing her PhD in chemistry at the Free University Berlin, she has been working in the nanoparticle technology department at the Fraunhofer Institute ICT-IMM in Mainz. Recently she was awarded a Fraunhofer Talenta grant and has started to build up a new group in the field of nanotherapeutica.

Raphael Thiermann received his degree in Chemistry from the University of Mainz in 2009. In Berlin he investigated the selfassembly of block copolymers in micromixers at the Federal Institute of Materials Research and Testing under the supervision of Prof. Maskos and obtained his PhD at the Technical University of Berlin in collaboration with Prof. Gradzielski. Since 2014 he works at the Fraunhofer Institute ICT-IMM in Mainz in the Nanoanalytics Group. His current research interests include self-assembly of amphiphiles, hybrid nanoparticles, surface modifications, continuous synthesis of nanoparticles, and characterization of nanomaterials. K


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(8) Mueller, W.; Koynov, K.; Fischer, K.; Hartmann, S.; Pierrat, S.; Basché, T.; Maskos, M. Macromolecules 2009, 42 (1), 357−361. (9) Mueller, W.; Koynov, K.; Pierrat, S.; Thiermann, R.; Fischer, K.; Maskos, M. Polymer 2011, 52 (5), 1263−1267. (10) Lammers, T.; Aime, S.; Hennink, W. E.; Storm, G.; Kiessling, F. Acc. Chem. Res. 2011, 44 (10), 1029−1038. (11) Choi, K. Y.; Liu, G.; Lee, S.; Chen, X. Nanoscale 2012, 4 (2), 330−342. (12) Pawar, P. V.; Gohil, S. V.; Jain, J. P.; Kumar, N. Polym. Chem. 2013, 4 (11), 3160−3176. (13) Sanson, C.; Diou, O.; Thévenot, J.; Ibarboure, E.; Soum, A.; Brûlet, A.; Miraux, S.; Thiaudière, E.; Tan, S.; Brisson, A.; Dupuis, V.; Sandre, O.; Lecommandoux, S. ACS Nano 2011, 5 (2), 1122−1140. (14) Meng, F. H.; Zhong, Z. Y.; Feijen, J. Biomacromolecules 2009, 10 (2), 197−209. (15) Langowska, K.; Palivan, C. G.; Meier, W. Chem. Commun. 2013, 49 (2), 128−130. (16) Meier, W.; Nardin, C.; Winterhalter, M. Angew. Chem., Int. Ed. 2000, 39, 4599. (17) Egli, S.; Nussbaumer, M. G.; Balasubramanian, V.; Chami, M.; Bruns, N.; Palivan, C.; Meier, W. J. Am. Chem. Soc. 2011, 133 (12), 4476−4483. (18) Egli, S.; Schlaad, H.; Bruns, N.; Meier, W. Polymers 2011, 3 (1), 252−280. (19) Opsteen, J. A.; Brinkhuis, R. P.; Teeuwen, R. L. M.; Loewik, D. W. P. M.; van Hest, J. C. M. Chem. Commun. 2007, 3136−3138. (20) Lee, J. C. M.; Bermudez, H.; Discher, B. M.; Sheehan, M. A.; Won, Y. Y.; Bates, F. S.; Discher, D. E. Biotechnol. Bioeng. 2001, 73 (2), 135−145. (21) Huang, J.; Bonduelle, C.; Thevenot, J.; Lecommandoux, S.; Heise, A. J. Am. Chem. Soc. 2012, 134 (1), 119−122. (22) Christian, N. A.; Milone, M. C.; Ranka, S. S.; Li, G.; Frail, P. R.; Davis, K. P.; Bates, F. S.; Therien, M. J.; Ghoroghchian, P. P.; June, C. H.; Hammer, D. A. Bioconjugate Chem. 2007, 18 (1), 31−40. (23) Lomas, H.; Canton, I.; MacNeil, S.; Du, J.; Armes, S. P.; Ryan, A. J.; Lewis, A. L.; Battaglia, G. Adv. Mater. 2007, 19 (23), 4238. (24) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. J. Controlled Release 2000, 65 (1−2), 271−284. (25) Azzopardi, E. A.; Ferguson, E. L.; Thomas, D. W. J. Antimicrob. Chemother. 2013, 68 (2), 257−274. (26) Tenzer, S.; Docter, D.; Rosfa, S.; Wlodarski, A.; Kuharev, J.; Rekik, A.; Knauer, S. K.; Bantz, C.; Nawroth, T.; Bier, C.; Sirirattanapan, J.; Mann, W.; Treuel, L.; Zellner, R.; Maskos, M.; Schild, H.; Stauber, R. H. ACS Nano 2011, 5 (9), 7155−7167. (27) Owens, D. E.; Peppas, N. A. Int. J. Pharm. 2006, 307 (1), 93− 102. (28) Brinkhuis, R. P.; Stojanov, K.; Laverman, P.; Eilander, J.; Zuhorn, I. S.; Rutjes, F. P. J. T.; van Hest, J. C. M. Bioconjugate Chem. 2012, 23 (5), 958−965. (29) Lankveld, D. P. K.; Oomen, A. G.; Krystek, P.; Neigh, A.; Troost-de Jong, A.; Noorlander, C. W.; Van Eijkeren, J. C. H.; Geertsma, R. E.; De Jong, W. H. Biomaterials 2010, 31 (32), 8350− 8361. (30) Litzinger, D. C.; Buiting, A. M. J.; van Rooijen, N.; Huang, L. Biochim. Biophys. Acta, Biomembr. 1994, 1190 (1), 99−107. (31) Harashima, H.; Kiwada, H. Adv. Drug Delivery Rev. 1996, 19 (3), 425−444. (32) Bertrand, N.; Leroux, J.-C. J. Controlled Release 2012, 161 (2), 152−163. (33) Wisse, E.; Jacobs, F.; Topal, B.; Frederik, P.; De Geest, B. Gene Ther. 2008, 15 (17), 1193−1199. (34) Shilo, M.; Sharon, A.; Baranes, K.; Motiei, M.; Lellouche, J.-P.; Popovtzer, R. J. Nanobiotechnol. 2015, 13 (1), 19. (35) Wong, A. D.; Ye, M.; Levy, A. F.; Rothstein, J. D.; Bergles, D. E.; Searson, P. C. Front. Neuroeng. 2013, 6, 7. (36) Lee, J. S.; Feijen, J. J. Controlled Release 2012, 161 (2), 473−483. (37) Leibler, L. Macromolecules 1980, 13 (6), 1602−1617. (38) Discher, D. E.; Eisenberg, A. Science 2002, 297 (5583), 967− 973.

Michael Maskos, graduate chemist, heads the Fraunhofer ICT-IMM as a director since 2014. Before that, he was CEO of the Institut für Mikrotechnik Mainz GmbH (IMM) since 2011, which was then integrated into the Fraunhofer-Gesellschaft in 2014. After his PhD at the Institute for Polymers of the University of Marburg, he worked five years as a scientific assistant at the Johannes-Gutenberg University in Mainz. In 2000, he received the Research Award of the BoehringerIngelheim Foundation. Afterwards he left Mainz for a year abroad within a research scholarchip of the German Academy of Natural Scientists Leopoldina. He worked as a visiting scientist at the McGill University in Montreal, Canada. Back in Germany Maskos finished his habilitation (physical chemistry) in 2003 and remained at the Johannes-Gutenberg University as assistant professor. In 2009, he moved to the German capital Berlin where he took the lead as director and professor of the Division Durability of Polymers at the Federal Institute of Materials Research and Testing (BAM). During this period he also graduated at the Helmholtz-Academy of Young Researchers in Scientific Management. Two years later he was appointed full professor at the Johannes-Gutenberg University in the field of Chemical Process Engineering/Microfluidics. Maskos came back to Mainz and became CEO of the IMM. In 2015, Maskos together with two colleagues won the Literature Prize of the Fonds der Chemischen Industrie for the textbook “Polymers: Synthesis, Characteristics and Applications”.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the European Regional Development Fund (EFRE). We also appreciate the support for this work from the Federal Institute for Material Science and Testing (BAM). R.B. thanks the German Academic Exchange Service (DAAD) for a PhD fellowship DAAD-PKZ: D/11/43560. We also thank Sibylle von Bomhard and Johanna Ringeisen for their kind assistance with the artwork.

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Techniques To Control Polymersome Size - Macromolecules (ACS

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