Tuning the membrane permeability of polymersome nanoreactors developed by aqueous emulsion polymerization-induced self-assembly

Polymeric vesicles (or polymersomes) are hollow bilayer structures consisting of an inner aqueous compartment enclosed by a hydrophobic membrane. Vesicular constructs are ubiquitous in nature and perform a variety of functions by compartmentalizing molecules into disparate environments. For polymer chemists, the synthesis of vesicles can be readily accomplished using polymerization-induced self-assembly (PISA), whereby pure vesicle morphologies can be easily accessed by tuning initial reaction parameters. Research into polymersomes is motivated primarily by the fact that hydrophilic cargo such as drug molecules, DNA, or enzymes can be encapsulated and protected from the often harsh conditions of the surrounding environment. A key factor governing the capability of vesicles to retain and protect their cargo is the permeability of their hydrophobic membrane. Herein, we demonstrate that membrane permeability of enzyme-loaded epoxy-functionalized polymersomes synthesized by aqueous emulsion PISA can be modulated via epoxide ring-opening with various diamine cross-linkers and hydrophobic primary amines. In general, membrane cross-linking or amine conjugation resulted in increased polymersome membrane thickness. Membrane modi ﬁ cation was also found to decrease permeability in all cases, as measured by enzymatically-catalysed oxidation of an externally administered substrate. Functionalization with hydrophobic amines resulted in the largest reduction in enzyme activity, suggesting signi ﬁ cant blocking of substrate di ﬀ usion into the central aqueous compartment. This procedurally facile strategy yields meaningful insight into how the chemical structure of the membrane in ﬂ uences permeability and thus could be generally applied to the formulation of polymeric vesicles for therapeutic applications.


Introduction
Structural organization is an essential feature of nature's toolbox for maintaining all forms of life.Evolution of compartmentalized environments on both cellular and subcellular level (i.e.organelles) allows for vital biological reactions to occur selectively in confined spaces that simultaneously separate and protect them from external detrimental agents. 1,2][4] Inspired by nature, researchers have developed methodologies to design minimal synthetic analogues that mimic these complex systems. 5][23][24][25][26] In particular, development of single-phase block copolymer vesicles via PISA in dispersed aqueous media has been primarily achieved using reversible addition-fragmentation chaintransfer (RAFT) polymerization, 22,27,28 as well as non-radical methodologies such as ring-opening metathesis polymerization (ROMP). 29,30However, a limited number of reports centered upon polymersomes prepared under emulsion PISA conditions have been introduced in the literature thus far, [31][32][33][34] while the factors that allow for higher-order morphologies other than kinetically trapped spheres to be accessed under emulsion polymerization conditions remain currently unclear.
6][37][38][39][40][41] Aqueous photo-PISA has enabled the direct non-disruptive encapsulation of inorganic nanoparticles, 36,42 as well as other sensitive (bio)molecules, such as fluorophores, 36,43 and proteins/enzymes, [43][44][45][46][47][48] into polymeric vesicles for the efficient construction of delivery vehicles, therapeutics, and catalytic nanoreactors with biologically relevant applications. 491][52][53][54] The membranes of such nanoconstructs were rendered permeable toward substrate molecules and catalysis products upon incorporation of channel-forming transmembrane proteins, 52,55 DNA nanopores, 56 or stimuli-responsive moieties 51,54 into their hydrophobic domains.Furthermore, a few reports were based upon the inherent permeability of the polymersome membranes for passive diffusion of small molecules between their outer and inner aqueous compartments. 57,58ore recently, our group has demonstrated the preparation of enzyme-loaded poly(ethylene glycol)-b-poly(2-hydroxypropyl methacrylate) (PEG-b-PHPMA) nanoreactors via one-pot aqueous photo-PISA that were able to communicate through their inherently permeable and relatively hydrated membranes, allowing for catalytic cascade reactions to occur inside separated compartments. 43Moreover, encapsulation of a therapeutic enzyme into the same system resulted in nanoreactors intended for leukemia treatment, whilst protection of the cargo from antibody binding and proteolytic degradation owing to the size-selective permeability of the PHPMA membrane was also presented. 46In a different study by our group, incorporation of a channel-forming porin protein into the membrane of PEG-b-PHPMA vesicles led to a significant permeability enhancement. 47Other attempts to enhance the membrane permeability of PISA polymersomes by incorporating pH-responsive units into their core-forming blocks for drug release applications have also been reported recently. 59,60erein, enzyme-loaded epoxy-functionalized polymersome nanoreactors of well-defined characteristics and inherent permeability were developed via aqueous RAFT-mediated emulsion photo-PISA at mild temperature using a mixture of 2-hydroxypropyl methacrylate (HPMA) and glycidyl methacrylate (GlyMA) as the core-forming monomers.2][63] In all cases, modification of the chemical composition of the core-forming block resulted in a distinct increase of vesicular membrane thickness and as a consequence in less-hydrated nanoreactors with tunable permeability toward small molecule substrates, as determined by enzymatic assays.Enhanced blocking efficiency was evident upon increasing the hydrophobicity of the nucleophile employed, allowing for identification of valuable structure-property relationships.Overall, our findings expand the current knowledge on membrane characteristics of semi-permeable nanocompartments and could facilitate the design of biomembrane-mimicking nanostructures and artificial "nanofactories" with programmed size-selective permeability via one-pot PISA.

Synthetic procedures
Synthesis of poly(ethylene glycol) 113 -based macromolecular chain transfer agent (PEG 113 macro-CTA).The synthesis of PEG 113 macro-CTA by N,N′-dicyclohexylcarbodiimide (DCC) coupling between poly(ethylene glycol) monomethyl ether (PEG 113 -OH) and 4-cyano-4-[(ethylsulfanylthiocarbonyl)sulfanyl] pentanoic acid (CEPA) was performed according to previously reported experimental protocols by our group and others. 36,40ynthesis of PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) diblock copolymer vesicles by aqueous RAFT-mediated emulsion photoinitiated polymerization-induced self-assembly (photo-PISA).Photo-PISA reactions were performed in a custom-built photoreactor setup.This ensured the polymerization solutions were only exposed to the light from the 400-410 nm LED source placed underneath the vials.The detailed description of the photoreactor setup specifications is given in the ESI.† A typical synthetic procedure to achieve epoxy-functionalized PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) diblock copolymer vesicles at [solids] = 10% w/w via aqueous RAFT-mediated emul-sion photo-PISA is described (Fig. 1A). 43,47PEG 113 macro-CTA (9.1 mg, 1.7 × 10 −6 mol, 1 eq.),HPMA (79 mg, 5.5 × 10 −4 mol, 320 eq.) (80 mol%) and GlyMA (19.9 mg, 1.4 × 10 −4 mol, 80 eq.) (20 mol%) were dispersed in deionized (DI) water (0.9 mL) with vigorous agitation using a vortex mixer for 2 min in a sealed 15 mL scintillation vial bearing a magnetic stirrer bar.The resulting monomer-in-water emulsion solution was degassed by sparging with N 2 (g) for 15 min.The sealed vial was incubated at 37 °C with magnetic stirring under 405 nm light irradiation for 2 hours to ensure full monomer conversion.After this period, the reaction mixture was exposed to air and allowed to cool to room temperature.FT-IR, 1 H-NMR in methanol-d 4 and SEC analyses in DMF + 5 mM NH 4 BF 4 of the pure diblock copolymers were performed after lyophilization of an aliquot of particles.DLS analysis and dry-state and cryo-TEM imaging were performed on samples after dilution to an appropriate analysis concentration.Encapsulation of horseradish peroxidase (HRP) into PEG 113b-P(HPMA 320 -co-GlyMA 80 ) vesicles by one-pot aqueous RAFTmediated emulsion photo-PISA.For the preparation of HRPloaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) nanoreactors by aqueous emulsion photo-PISA at 10% w/w solids content, a typical synthetic protocol was followed. 43PEG 113 macro-CTA (9.1 mg, 1.7 × 10 −6 mol, 1 eq.),HPMA (79 mg, 5.5 × 10 −4 mol, 320 eq.) (80 mol%) and GlyMA (19.9 mg, 1.4 × 10 −4 mol, 80 eq.) (20 mol%) were dispersed in DI water (0.8 mL) with vigorous agitation using a vortex mixer for 2 min in a sealed 15 mL scintillation vial bearing a magnetic stirrer bar.Then, 0.1 mL of a 200 U mL −1 HRP solution in DI water was added.The resulting emulsion solution was degassed by sparging with N 2 (g) for 15 min.The sealed vial was incubated at 37 °C with magnetic stirring under 405 nm light irradiation for 2 hours to ensure full monomer conversion.After this period, the reaction mixture was exposed to air and allowed to cool to room temperature.The resulting solution of HRP-loaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) vesicles was then diluted 10-fold in DI water or 100 mM PB ( pH = 5.5, PB 5.5) and purified, respectively, by three centrifugation/resuspension cycles in DI water or 100 mM PB 5.5 at 14 000 rpm for the removal of unreacted monomer and free HRP enzyme.
For Michaelis-Menten kinetics determination, activity of purified HRP-loaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) vesicles before and after post-PISA membrane functionalization using BA, NMA, and PXDA was evaluated at different [DMB] (0-6 mM) with H 2 O 2 under saturating conditions.Vesicles at 20× dilution (0.5% w/w solids content) in 100 mM PB 5.5 (120 μL) were diluted with 100 mM PB 5.5 (20 μL) in a 96-well plate microwell.DMB of appropriate concentration (0-30 mM, 40 μL) was then added.Finally, a 35% w/v aqueous solution of hydrogen peroxide (20 μL) was added, and the change in absorbance was monitored in an identical manner.Absorbance values were corrected against particle absorbance at t = 0 min and reported as ΔAbs 492 nm .In each case, the average initial slope of three repeat measurements (V 0 ) for the first 10 min of the assay was used for construction of Michaelis-Menten kinetic plots and was normalized against V max (Table S3 †).Calculated K * m values are presented as mean ± standard error.
A similar process was followed for activity testing of the free enzyme after a series of control experiments at appropriate [HRP].The free HRP solutions at final [HRP] = 1 U mL −1 in 100 mM PB 5.5 (20 μL) were diluted with 100 mM PB 5.5 (120 μL) in a 96-well plate microwell.DMB (2 mM, 40 μL) was then added.Finally, a 35% w/v aqueous solution of hydrogen peroxide (20 μL) was added, and the change in absorbance was monitored in an identical manner.In all cases, measurements were performed in at least triplicate and results are reported as their average values.

Results and discussion
Synthesis of epoxy-functionalized PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) polymersomes via aqueous RAFT-mediated emulsion photo-PISA Following related PISA studies previously reported by our group and others, 36,40 a water-soluble poly(ethylene glycol) macromolecular chain transfer agent (PEG 113 macro-CTA) was first synthesized through an esterification reaction between poly(ethylene glycol) monomethyl ether (PEG 113 -OH) and an acid-functionalized CTA (esterification efficiency = 93%, M n, SEC = 12.4 kg mol −1 , Đ M = 1.05).Aqueous RAFT-mediated photoinitiated PISA ( photo-PISA) of a mixture of commercially available water-miscible 2-hydroxypropyl methacrylate (HPMA) and water-immiscible glycidyl methacrylate (GlyMA) as the core-forming monomers was achieved under emulsion polymerization conditions, using PEG 113 macro-CTA as both the steric stabilizer block and the surfactant for stabilization of the heterogeneous monomer-in-water solution. 64Dispersed monomer droplets of varying size (5-30 μm) were observed upon optical microscopy imaging of the formed emulsion solution prior to polymerization (Fig. S2 †).
Single-phase epoxy-functionalized PEG 113 -b-P(HPMA-co-GlyMA) diblock copolymer vesicles were developed upon irradiation of the polymerization solution under 405 nm visible light in the absence of an externally added photoinitiator or catalyst ("photoiniferter" mechanism of trithiocarbonates) 35,65,66 at 37 °C, targeting DP PHPMA = 320 (80 mol%) and DP PGlyMA = 80 (20 mol%) at 10% w/w solids content (Fig. 1A).Emulsion photo-PISA was carried out at mild temperature to ensure quantitative retention of epoxy functional groups, as epoxides can undergo partial hydrolysis in aqueous solution at elevated temperatures (60-100 °C). 61mportantly, 99% of PGlyMA pendant epoxy groups remained intact after photo-PISA, as calculated from 1 H-NMR spectroscopy by comparing the integral ratio of the peaks corresponding to epoxy proton signals at 2.73 and 2.91 ppm (I 2.73/2.91 ppm = 1.98) to the peak of the methacrylic backbone -CH 2protons at 1.50-2.30ppm (I 1.50-2.30ppm = 10.00) (Fig. S3 †).A kinetic study revealed that near complete monomer conversion (>98%) was achieved after 2 hours of irradiation time, as calculated by 1 H-NMR spectroscopy, whilst the onset of particle micellization accompanied by an increase in polymerization rate was observed after ∼20 min of irradiation time (ca.17% monomer conversion) (Fig. 1B and S4A †).The distribution of PGlyMA units along the growing polymer chain was also investigated during kinetics monitoring and the calculated HPMA/GlyMA molar ratio varied between 3 and 4 throughout the whole duration of photo-PISA, suggesting the formation of random copolymers (Fig. S4B †).The prepared PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) diblock copolymer possessed monomodal molecular weight distribution with relatively low dispersity value, as determined by SEC analysis (M n, SEC = 87.9kg mol −1 , Đ M = 1.43) (Fig. 1C).
Dynamic light scattering (DLS) analysis revealed the formation of nano-objects with a monomodal size distribution and mean hydrodynamic diameter (D h ) of 188.3 ± 5.6 nm, low polydispersity (PD = 0.18 ± 0.02) and a smooth, single exponential decay autocorrelation function with optimal signal-tonoise ratio (Y-intercept >0.9) (Fig. 2A and B).The absence of charges on the outer surface of the developed particles was confirmed by microelectrophoretic analysis at neutral pH (measured zeta-potential = −1.87 ± 0.10 mV).Dry-state transmission electron microscopy (TEM) imaging confirmed the successful development of well-defined PEG 113 -b-P(HPMA 320co-GlyMA 80 ) unilamellar vesicles of spherical shape and uniform size (D dry-state = 170.3± 23.0 nm) (Fig. 2C and S5A †).Additionally, the characteristics of PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) vesicles in solution were studied by cryogenic TEM (cryo-TEM) imaging.Average vesicle size measured from particle counting analysis based on cryo-TEM was in good agree-ment with values determined by DLS and dry-state TEM analyses (D cryo = 175.5 ± 24.1 nm), while calculated average membrane thickness was M ave = 28.0 ± 3.0 nm (Fig. 2D-F

and S5B †).
Preparation of catalytic enzyme-loaded PEG 113 -b-P(HPMA 320co-GlyMA 80 ) polymersome nanoreactors via one-pot aqueous emulsion photo-PISA Next, PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) polymersomes were used as a nanocarrier platform for loading of a model hydrophilic enzyme to prepare catalytically active epoxy-functionalized nanoreactors. 43,47Enzyme horseradish peroxidase (HRP) was encapsulated into the inner aqueous compartment of the vesicles by performing a one-pot emulsion photo-PISA process under the same mild reaction conditions described herein in the presence of an aqueous HRP solution for the development of enzyme-loaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) polymersome nanoreactors.Purification of the vesicle sample for complete removal of free HRP was achieved by consecutive centrifugation/resuspension cycles in either deionized (DI) water or 100 mM phosphate buffer ( pH = 5.5, PB 5.5).
Importantly, the encapsulation process of HRP into PEG 113b-P(HPMA 320 -co-GlyMA 80 ) polymersomes did not alter their overall characteristics as judged by DLS analysis and TEM imaging (Fig. S6 †).Comparable D h and PD values were measured for HRP-loaded vesicles with respect to non-HRPloaded vesicles (D h = 182.6 ± 2.5 nm, and PD = 0.14 ± 0.04), whilst average vesicle size and average membrane thickness measured from cryo-TEM images were D cryo = 172.5 ± 25.5 nm and M ave = 27.9 ± 2.6 nm, respectively.
HRP is known to catalyze the oxidation reaction of colourless substrate 3,3′-dimethoxybenzidine (DMB) to a coloured dimer product (red-brown), which can be monitored by measuring the change in absorbance at λ = 492 nm over time via kinetic colorimetric analysis.This assay provides a read-out of enzyme activity and consequently of polymersome membrane permeability.Notably, PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) vesicles were found to be inherently permeable toward small molecule DMB, although they presented reduced enzyme activity compared to their reported PEG 113 -b-PHPMA 400 counterparts presumably due to increased membrane hydrophobicity in the former case. 43Although nanoreactors with near identical membrane thickness values were developed in both cases, the presence of an additional hydrophobic coreblock component in the case of PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) vesicles resulted in particles with less hydrated membranes and reduced permeability, making the exchange of substrates between their outer and inner compartment a more difficult process. 47Control experiments to assess the activity of free HRP after incubation at 37 °C for 2 hours in 10% w/w HPMA or HPMA/GlyMA (80 : 20 molar ratio) monomer solutions in DI water under 405 nm irradiation ( photo-PISA conditions) revealed quantitative retention of activity in both cases compared to untreated enzyme, showing that the enzyme function was not affected by either monomer (Fig. S7 †).In addition, high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) were employed to determine potential modification of HRP by ring-opening of the epoxide groups via its lysine residues.Notably, incubation of free HRP with a water-soluble small molecule epoxide (glycidol) for 2 hours in DI water showed no evident modification upon comparison with the untreated enzyme (Fig. S8 †).
Membrane functionalization of enzyme-loaded PEG 113 -b-P (HPMA 320 -co-GlyMA 80 ) vesicles via ring-opening of epoxide groups using primary amines Incorporation of PGlyMA units and thus the presence of pendant epoxy groups within the hydrophobic membrane of prepared HRP-loaded nanoreactors provides a reactive handle that allows for post-PISA functionalization of the polymersomes with nucleophilic compounds.2][63] To this extent, we envisioned that introducing hydrophobic or crosslinking moieties into the polymersome membrane would result in permeability reduction or nanoreactors completely impermeable toward small molecules.
A series of commercially available primary amines and diamines (cross-linking agents) of varying hydrophobicity were chosen as nucleophiles for ring-opening of PGlyMA epoxy groups and subsequent vesicle membrane modification (Scheme 1).Post-polymerization functionalization was achieved by mixing purified HRP-loaded PEG 113 -b-P(HPMA 320co-GlyMA 80 ) vesicle solutions in DI water at [solids] = 1% w/w with different amines at room temperature for 18 hours.In all cases, the [amine]/[epoxide] molar ratio was constantly maintained at 2.0 (i.e.2][63] Following this period, membrane-functionalized polymersomes were trans-ferred into PB 5.5 (optimum pH value of HRP) by one centrifugation/resuspension cycle prior to kinetic colorimetric analysis for enzyme activity and nanoreactor permeability determination.FT-IR spectroscopy was used for the confirmation of successful ring-opening of PGlyMA units in each case by monitoring the characteristic asymmetric vibration peaks of epoxide groups at 849 and 909 cm −1 before and after ring-opening reactions (Fig. S11, S14 and S16 †).It should also be noted that incubation of free HRP in DI water at room temperature for 18 hours as a control experiment resulted in no loss of enzyme activity (>99% retention of activity), compared to incubation of the enzyme in PB 5.5 for the same period of time (Fig. S9 †).Additionally, a control experiment for comparison of the activity of non-epoxy-functionalized and purified HRP-loaded PEG 113 -b-PHPMA 400 vesicles before and after incubation with the utilized primary amine and diamine molecules at room temperature for 18 hours in DI water, highlighted that the prolonged presence of amines in the particle dispersion solutions did not affect the catalytic activity of HRP (>95% retention of activity in all casescontrol vesicle solutions after incubation with C n DA cross-linkers could not be resuspended in PB 5.5 and hence their activity was not assessed) (Fig. S10 †).
Ring-opening of PGlyMA units using linear poly(ethylene glycol) diamines (PEG n DA) as cross-linkers.First, two watersoluble poly(ethylene glycol)-based diamine polymers of differing molecular weight (PEG n DA, n = 23 or 46) were selected as cross-linking agents for post-PISA membrane functionalization of HRP-loaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) nanoreactors following the above described process (Scheme 1).In both cases, efficient ring-opening of epoxy groups of PGlyMA using polymeric PEG 23/46 DA as cross-linkers was confirmed by FT-IR spectroscopy (Fig. S11 †) that resulted in a noticeable increase of nanostructure size.D h values of PEG n DA-modified particles were approximately 240 nm (PD = 0.11-0.14),as measured by DLS analysis (Fig. 3A-I and B-I), whereas the hydrodynamic diameter of the original PEG 113 -b-P (HPMA 320 -co-GlyMA 80 ) polymersome platform was ∼180 nm.No evident macroscopic precipitation of the particle solutions was observed that could imply inter-vesicular cross-linking.In particular, dry-state and cryo-TEM imaging revealed the retention of vesicular morphology in both cases with no apparent particle agglomeration, whilst average vesicle sizes determined from particle counting analysis based on cryo-TEM images were in excellent agreement with DLS results (D cryo, PEG 23 DA = 224.0± 21.0 nm, and D cryo, PEG 46 DA = 218.5 ± 22.3 nm) (Fig. 3A-II, B-II and S12 †).The observed size increase can be explained by the hydrophilic nature of the PEG n DA crosslinkers that could potentially lead to hydration of the vesicle membranes and as a result to partial swelling of the assemblies.Moreover, a measurable increase in average membrane thickness to ∼32 nm was noticed in both cases compared to the non-functionalized vesicles (Fig. 3A-III and B-III).
Surprisingly, activity comparison between encapsulated HRP into inherently permeable PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) vesicles and PEG n DA cross-linked PEG 113 -b-P (HPMA 320 -co-GlyMA 80 ) vesicles showed a significant enzyme activity decrease for the latter ones.More specifically, an absorbance decrease of 45 ± 5% was measured from kinetic colorimetric analysis after 30 min in the case of cross-linked PEG 113b-P(HPMA 320 -co-GlyMA 80 ) + PEG 23 DA vesicles compared to their non-functionalized epoxide-containing counterparts, while a further absorbance decrease of 69 ± 4% was identified upon increasing the molecular weight of the PEG n DA crosslinker (n = 46) (Fig. 3C and S13 †).The observed decrease in HRP activity demonstrates the permeability reduction of PEG n DA-functionalized nanoreactors toward DMB and hydrogen peroxide.Contrary to the observed particle swelling that would suggest a potential permeability enhancement, this can be understood in terms of the considerably thicker and more densely packed vesicular membranes after cross-linking that create an additional diffusive barrier which in turn hinders the passage of small molecule substrates to reach the active enzyme site. 43ing-opening of PGlyMA units using linear aliphatic diamines (C n DA) as cross-linkers.Next, a series of linear aliphatic diamine small molecules with increasing chain length (C n DA, n = 2, 3, 4, 6) were selected as cross-linkers for ring-opening of epoxy groups located in the hydrophobic domain of HRPloaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) vesicles (Scheme 1).We hypothesized that increasing the hydrophobicity of the cross-linking moiety compared to PEG n DA would result in a more pronounced enzyme activity reduction similar to the background activity (∼0%) of empty vesicles.This would allow for fabrication of nanoreactors that are effectively impermeable toward substrate molecules, whereby their encapsulated cargo is isolated from the outer aqueous surrounding environment of the particles.
FT-IR spectroscopy was used to confirm the successful modification of the polymersome membrane (i.e.dis- appearance of the characteristic bands of epoxide groups at 849 and 909 cm −1 ) (Fig. S14 †).Interestingly, DLS analysis of the C n DA-functionalized HRP-loaded polymersomes revealed a size decrease to 125-130 nm (PD = 0.12-0.13)for the shorter C 2 DA and C 3 DA cross-linkers compared to the original PEG 113b-P(HPMA 320 -co-GlyMA 80 ) vesicles which was attributed to shrinkage of the particles upon cross-linking with hydrophobic compounds that results in exclusion of water molecules from the membrane.In the case of longer C 4 DA and C 6 DA crosslinkers, a vast D h increase was measured to 1497 ± 266 nm and 1389 ± 149 nm, respectively, accompanied by a noticeable polydispersity increase (PD = 0.23-0.24)(Fig. 4A).These findings suggest the occurrence of particle agglomeration in the latter cases due to the development of inter-vesicular interactions resulting in the apparent population of particles with increased size.This was also evident by visual inspection of the PISA solutions upon functionalization after one week, where macroscopic precipitation was visible to some extent for PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) + C 4/6 DA assemblies, implying the occurrence of time-dependent coagulation in aqueous media.The morphology retention of HRP-loaded PEG 113 -b-P (HPMA 320 -co-GlyMA 80 ) + C n DA polymersomes was also verified by dry-state and cryo-TEM imaging after post-PISA functionalization (D cryo = 116-120 nm), for which near identical vesicle formulations were observed in all cases.A negligible population of aggregated particles could only be observed in the case of C 4 DA and C 6 DA-functionalized polymersomes that supports the DLS findings described above.The average membrane thickness of the prepared nanoreactors was also calculated from particle counting analysis in each case.Increased membrane thickness values of M ave = 30-32 nm were measured in all cases, comparable to PEG n DA-functionalized vesicles (Fig. 4B and S15 †).
Similar to PEG n DA cross-linked vesicles, assessment of the enzyme activity of HRP-loaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) + C n DA nanoreactors through kinetic colorimetric analysis revealed an absorbance decrease ranging from 50% to 61% depending on the cross-linker molecule used as compared to their PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) counterparts (Fig. 4C).The absorbance decrease measured was approximately the same in all cases, whilst there was no apparent trend based on the length of the cross-linker used for ringopening of PGlyMA units (i.e.increasing the C n DA length didn't result in an analogous HRP activity decrease).Overall, membrane cross-linking of the vesicles using C n DA molecules and subsequent introduction of additional hydrophobicity yielded particles with thicker and possibly less hydrated membranes with markedly reduced permeability.
Ring-opening of PGlyMA units using other hydrophobic primary amines as nucleophiles.As a next step, a more hydrophobic cross-linking xylylene-based diamine (PXDA) was selected for ring-opening of PGlyMA epoxy groups along with three other hydrophobic compounds bearing a single amino group in their structure (i.e.BA, NMA, and AMA) to prevent formation of linkages and agglomeration of the vesicles, as observed for longer C n DA cross-linkers, and to achieve com- plete reduction of membrane permeability for enzyme-loaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) polymersome nanoreactors (Scheme 1).Notably, the majority of selected amines resulted in fabrication of stable nanoparticle dispersions, except for AMA, where prominent macroscopic precipitation and formation of a film at the bottom of the vial was observed after epoxide ring-opening reaction, implying instability of the nano-objects in this case due to significantly increased hydrophobicity.In all cases, FT-IR spectroscopic analysis confirmed the successful ring-opening of PGlyMA epoxide groups (Fig. S16 †).
In the case of BA-functionalized vesicles, D h and PD values comparable with those of the original HRP-loaded vesicles were measured from DLS analysis (D h = 203.4± 2.8 nm and PD = 0.08) (Fig. 5A-I and II).Dry-state TEM imaging was first used to confirm that post-PISA functionalization did not alter their morphology and uniform size (Fig. 5A-III and S17A-I †), while size and membrane thickness measurements were carried out from particle counting analysis based on cryo-TEM images (Fig. 5A-IV to VI and S17A-II †).Importantly, the average diameter of BA membrane-modified vesicles was in good agreement with DLS results (D cryo, BA = 175.4± 27.7 nm), whereas a significant increase in average membrane thickness was also observed (M ave, BA = 33.2± 2.9 nm) compared to PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) vesicles.
Near identical well-defined vesicle formulations of low PD and similar D h values were identified by DLS analysis and drystate TEM imaging in the case of NMA and PXDA amines (Fig. 5B-I to III, C-I to III and S17 †).Similar to HRP-loaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) + BA nanoreactors, average D cryo values were in good agreement with light scattering results, whilst an increased average membrane thickness was calculated for PXDA-functionalized vesicles (M ave, PXDA = 31.1 ± 2.7 nm).Additionally, a further membrane thickness increase to M ave, NMA = 37.6 ± 2.8 nm was calculated upon PGlyMA ringopening using a more hydrophobic amine (i.e.NMA), showing an evident relationship between the structure/hydrophobicity of the nucleophile and the membrane thickness of the resulting vesicles (Fig. 5B-IV to VI, C-IV to VI and S17 †).
The activity of HRP-loaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) + BA/NMA/PXDA polymersomes was assessed by kinetic colorimetric analysis, monitoring the enzyme-catalyzed peroxidation of DMB in line with above described assays (Fig. 6A).The rate of DMB peroxidation was clearly affected by the physicochemical nature of the polymersome ( particularly M ave ), with reductions in substrate processing correlating to increasing thickness of the diffusional barriers provided by the functionalized membrane.At a fixed substrate concentration (0.4 mM DMB and 1.1 M H 2 O 2 ), there was a small reduction in product formation for the PXDA cross-linked polymersomes of 31 ± 4% upon comparison with the original HRP-loaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) vesicles that dropped to >80% in the case of BA and NMA-functionalized nanoreactors (Fig. 6B).Although these values correlate well with measured M ave values (i.e.NMA > BA > PXDA > PGlyMA) and showcase how post-PISA chemical modification affects the membrane character altering its relative permeability, it was important to expand this data to explore the effect of substrate concentration; measuring the observable permeability.No change to the actual K m of the intact enzyme was taken into consider- ation as such fundamental changes to activity are not generally associated with encapsulation. 67Normalized kinetic data was presented in terms of V 0 /V max due to the focus on comparisons between equilibrium substrate dissociation constant (K m ) of the nanoreactor-loaded enzyme rather than absolute activity; an [enzyme]-dependent parameter.
Systematically varying [DMB] (with [H 2 O 2 ] under saturating conditions) gave a more detailed insight into the effect of membrane modification on the performance of nanoreactors, where the trend identified previously was confirmed and could be elucidated with greater detail (Fig. 6C-I and S18 †).As compared to the K m value of the free enzyme at 0.79 ± 0.06 mM, the K * m of HRP-loaded PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) nanoreactors increased by ca.30% (up to 1.14 ± 0.18 mM) as a result of enzyme compartmentalization within the semi-permeable P(HPMA 320 -co-GlyMA 80 ) membrane.This effect could be tuned by introducing cross-linking or chemical modification.PXDA cross-linking of polymersomes further increased K * m by ca.30% (up to 1.84 ± 0.18 mM), in accordance with earlier data under fixed substrate concentration conditions.Interestingly, using such kinetic assays, we were able to resolve the effect of BA and NMA modification on the permeability of the polymersome membrane.Indeed, post-PISA membrane modification and insertion of benzylic (BA) groups gave rise to a 70% increase in K * m , which increased by a further 230% when using bulkier naphthyl (NMA) groups (up to 3.14 ± 0.32 & 10.27 ± 4.34 mM, respectively).Overall, the observed rate of substrate turnover of the unmodified polymersomes was ca.90% higher than that of the NMA-functionalized nanoreactors.Relating these values for K * m to the measured values of average membrane thickness (M ave ), it is apparent that their relationship is non-linear, which means that the physicochemical origin of this tunability cannot be fully explicated by the influence of chemical modifier upon the membrane dimensions but is also related to the nature of the new membrane (Fig. 6C-II).In this instance, we can understand the increasing K * m value (and decreasing permeability) between BA and NMA membranes arising due to the increasing non-covalent aromatic interactions between polymer chains (i.e.increasing the density and reducing the porosity of the bilayer).
Overall, our findings provide a more in depth understanding of how relatively simple chemistries, used for the functionalization of polymersome membranes with increasingly more hydrophobic moieties, can tune the passive diffusion of small molecules into their aqueous inner lumen.This strategy has been utilized to achieve greater control over substrate processing by enzymes encapsulated within a polymersome nanoreactor.

Conclusions
In summary, we report a facile strategy to modulate the membrane permeability of polymeric vesicles.Epoxy-containing, enzyme-loaded vesicle nanoreactors obtained via aqueous emulsion PISA were functionalized with a series of diamine cross-linkers or hydrophobic primary amines using a simple procedure.Membrane modification resulted in increased thickness and reduced permeability relative to the non-functionalized particles.Of the compounds tested, the hydrophobic amines exhibited the most dramatic blocking effect on the vesicle membranes, reducing permeability by over 80% as determined by a colorimetric assay involving substrate oxidation by the encapsulated enzymes.This fundamental study reveals important insight into the relationship between membrane thickness, cross-linking density, or hydrophobicity and permeability.Such fine control over the diffusion of substrates across inherently permeable vesicular membranes has rarely been demonstrated and should inform particle design in future studies.

Fig. 2 (
Fig. 2 (A) Intensity-weighted size distributions along with average D h and PD values (the error shows the standard deviation from 5 repeat measurements), and (B) autocorrelation function obtained by DLS for empty PEG 113 -b-P(HPMA 320 -co-GlyMA 80 ) vesicles.(C) Representative dry-state TEM image, stained with 1 wt% uranyl acetate (UA) solution, and (D) representative cryo-TEM image of empty PEG 113 -b-P(HPMA 320co-GlyMA 80 ) vesicles.(E) Histogram of size distribution, and (F) histogram of membrane thickness distribution along with calculated average diameter and membrane thickness values, respectively, measured from particle analysis based on cryo-TEM images for empty PEG 113 -b-P (HPMA 320 -co-GlyMA 80 ) vesicles.In each case, at least 100 particles were analyzed.