Mixed films of 33:67 AB1 : OB18* were hydrated for the indicated times and vesicles were imaged at each time point to measure the domains per area (Avg ± S.E., n ≥ 3 per time point). The coarsening of domains is shown to saturate by ~40 hr, but the % Area occupied by domains remains relatively constant throughout. As shown, a majority of polymersomes showed fully segregated domains after 2 or more days. Scale bars = 2 µm.

Cation-induced, lateral phase segregation of charged AB1 and neutral, fluorescently-labeled OB18* diblock copolymers formed at pH₀ 4, 0.1 mM calcium at (a) 25% AB1, (b) 50% AB1, and (c) 75% AB1. (a, inset): Schematic of phase-separated membrane of thickness d. (d) Two color micrograph of a phase separated polymersome (34% AB1, AB1:AB1* = 10:1) with individual red (OB18*) and blue (AB1*) channel micrographs to illustrate the extent of demixing. (e) Individual channels modified using an averaging smoothing filter to enhance contrast for a two color phase separated polymersome (50% AB1) with 2% PIP2-BodipyFL (green) enriched in dark AB1 domains and partially segregated from the OB18*-rich domains (red). Unmodified images and fluorescence intensity analysis of domains are shown in Fig. S2. (f) Phase separation of AB1 (50%) induced by copper(II) at pH₀ 3.5, 0.075 mM copper(II). (g) Janus polymersome resulting from domain coarsening in a 50% AB1 polymersome.

(a) Time sequence of a pinned, striped worm-like cylinder micelle formed with AB1:OB18* = 25:75 at pH₀ 4.5 and [Ca²⁺] = 0.05 mM; the micelle remains flexible as evident in the contour overlay. (b) Striped cylinder micelles formed at increasing AB1 fraction, although for any sample only about 10% of cylinders appeared definitively striped with multiple micron-sized bands. Plots of thresholded fluorescence intensity along the worm contour length highlight average domain size and periodicity. (c) The long range ordering of a single 75:25 worm micelle is at least 17 µm as determined by examining the autocorrelation of the backbone fluorescence intensity. (d) Domain lengths (L) of OB18* in striped cylinders at varying AB1 fraction are fit to a Zimm-Schulz model (R² ≥ 0.95) in which the number average length, L_n*, scales linearly (R² = 0.96) with OB18* blend fraction (inset). Error bars represent bin sizes for L. Scale bar = 4 µm.

(a) Effective elasticity of AB1 polymersomes hydrated at the indicated Ca²⁺ and pH₀. Power law fits of the low strain regime provide a measure of Effective Rigidity E_app = (τ/x)^m in units of mN/m, which reveals a fluid, elastic membrane at pH₀ 4 (m = 1) and inelastic deformation of a gel-like membrane at pH₀ 6 (m = 1.25). Inset images respectively show spherical and non-spherical vesicle contours, consistent with fluid or solid-like behavior. (b, c) Power law fits of E_app as a function of pH or calcium quantify the stiffening, and much stronger effects with calcium. Fitting with Eq. 1 yields for [H⁺]: a_H+ = −0.34, A_H+ = 6.0 mN/m using a pK_a = 5 (R² = 0.99). For Ca²⁺, a_Ca = 2.5 and A_Ca = 0.002 mN/m (R² = 0.99). (d, e) Relative fluorescence intensity versus time from FRAP of cationic DiO:C18 dye in AB1 polymersomes at varying pH₀. Fits provide the characteristic diffusion times τ_D, and the calculated diffusion coefficients versus pH₀ are fit by a power law across the fluid-gel transition regime (solid line).

(a) Systematic experimental phase diagram of domain formation for 25% AB1 is overlayed on the morphological phase diagram for pure AB1 (shown in gray; spherical micelles (S), worm-like micelles (W), and vesicles (V)) and displayed with points where experiments indicate whether pure AB1 polymersomes (colored circles) and worm-like cylinder micelles (colored squares) display fluid (blue) or gel (orange) characteristics. The calcium values in the morphological phase diagram were recalculated to correspond to the 0.025 mg/mL AB1 present in the blends used for domain formation while pH was converted to [H⁺]. Four regions of interest were observed – buds, spots, squids, and stripes – as depicted in the inset images (scale bar = 2 µm). (b) The narrow window of domain formation observed experimentally is recapitulated using a theoretical model (solid line). Increasing blue intensity in the region of domains formation is representative of increasing contrast between laterally segregated domains, where the dash-dotted line bounds the region where the contrast is larger than 50%. The dominating physical mechanisms for mixing are indicated with text. (c) Contours for 50% contrast are plotted for other constituent ratios where increasing line thickness represents an increase in AB1 fraction (25%, 50%, 75%) for cylinder micelles (dotted) and polymersomes (dash-dotted). These contours are plotted over the region of experimentally observed domain formation. The high calcium boundary is not obtained because the theoretical model does not capture charge inversion.