Polymerization-Induced Polymersome Fusion

The dynamic interactions of membranes, particularly their fusion and fission, are critical for the transmission of chemical information between cells. Fusion is primarily driven by membrane tension built up through membrane deformation. For artificial polymersomes, fusion is commonly induced via the external application of a force field. Herein, fusion-promoted development of anisotropic tubular polymersomes (tubesomes) was achieved in the absence of an external force by exploiting the unique features of aqueous ring-opening metathesis polymerization-induced self-assembly (ROMPISA). The out-of-equilibrium tubesome morphology was found to arise spontaneously during polymerization, and the composition of each tubesome sample (purity and length distribution) could be manipulated simply by targeting different core-block degrees of polymerization (DPs). The evolution of tubesomes was shown to occur via fusion of “monomeric” spherical polymersomes, evidenced most notably by a step-growth-like relationship between the fraction of tubular to spherical nano-objects and the average number of fused particles per tubesome (analogous to monomer conversion and DP, respectively). Fusion was also confirmed by Förster resonance energy transfer (FRET) studies to show membrane blending and confocal microscopy imaging to show mixing of the polymersome lumens. We term this unique phenomenon polymerization-induced polymersome fusion, which operates via the buildup of membrane tension exerted by the growing polymer chains. Given the growing body of evidence demonstrating the importance of nanoparticle shape on biological activity, our methodology provides a facile route to reproducibly obtain samples containing mixtures of spherical and tubular polymersomes, or pure samples of tubesomes, of programmed length. Moreover, the capability to mix the interior aqueous compartments of polymersomes during polymerization-induced fusion also presents opportunities for its application in catalysis, small molecule trafficking, and drug delivery.


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High-Resolution Mass Spectrometry. HRMS spectra were recorded by the MS Analytical Facility Service at the University of Birmingham on a Waters Xevo G2-XS QTof Quadrupole Time-of-Flight mass spectrometer.
Size Exclusion Chromatography. Size exclusion chromatography (SEC) analysis was performed on a system composed of an Agilent 1260 Infinity II LC system equipped with an Agilent guard column (PLGel 5 μM, 50 × 7.5 mm) and two Agilent Mixed-C columns (PLGel 5 μM, 300 × 7.5 mm). The mobile phase used was THF (HPLC grade) containing 2% v/v NEt3 at 40 °C at flow rate of 1.0 mL min -1 (polystyrene (PS) standards were used for calibration). Detection was conducted using a differential refractive index Differential Scanning Calorimetry. Determination of the glass transition temperature (Tg) for P(NB-MEG)180 homopolymer was performed using a Mettler Toledo DSC 3 differential scanning calorimeter by heating the sample from 25 °C to 190 °C at a rate of 10 °C/min for two heating/cooling cycles. The Tg was determined from the inflection point in the second heating cycle of DSC. Collected data were processed using STARe software.
Turbidimetry. Turbidimetric analysis was performed on a Thermo Scientific Evolution™ 350 UV-Vis spectrophotometer equipped with a Peltier heating and cooling system. In situ ROMPISA kinetic analysis was performed by monitoring changes in transmittance of each polymerization solution at λ = 550 nm every 6 s over a period of 20 min at 25 °C. A solution of appropriately diluted P(NB-amine)11 or P(NB-PEG)11 macroinitiator in phosphate buffer at pH = 2.0 (PB 2) was used as a reference sample.
Dynamic Light Scattering. Hydrodynamic diameters (Dh) and size distributions (PD) of nano-objects were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS with a 4 mW He-Ne 633 nm laser module operating at 25 °C. Measurements were carried out at an angle of 173° (back scattering), and results were analyzed using Malvern DTS v7.03 software. All determinations were repeated 4 times with at least 10 measurements recorded for each run. Dh values were calculated using the Stokes-Einstein equation where particles are assumed to be spherical, while for cylindrical particles DLS was used to detect multiple populations and obtain dispersity information.
S4 Zeta Potential Analysis. Zeta potential was measured by the technique of microelectrophoresis, using a Malvern Zetasizer Nano ZS instrument, at room temperature at 633 nm. Owing to the high conductivity (>5 mS/cm) of the analysis media (phosphate buffer at pH = 2.0 with or without 100 mM NaCl), monomodal mode was selected for measurements using an applied voltage of 10 V. All reported zeta potential values were the average of at least three runs with at least 40 measurements recorded for each run. Zeta potential was calculated from the corresponding electrophoretic mobilities (μE) by using the Henry's correction of the Smoluchowski equation (μE = 4π ε0 εr ζ (1+κr)/6π μ).
Static Light Scattering. Static light scattering (SLS) analysis was performed using an ALV/CGS-3 compact goniometer system. 0.1 mg mL -1 solutions were filtered through 0.45 μm nylon filters prior to light scattering analysis at multiple angles ranging from 35° to 150° against a toluene standard. The wavelength of the incident beam was λ = 633 nm and for each angle, runs of at least 60 s were carried out at 20 °C. dn/dc values were determined using a DnDc1260 differential refractometer supplied by PSS GmbH (laser λ = 620 nm, Τ = 20 °C) at concentrations in the range 0.1 mg ml -1 ≤ c ≤ 0.5 mg ml -1 .
For each sample, the resulting g2(q, t) autocorrelation functions from DLS analysis for each angle were analyzed by the REPES algorithm to determine a relaxation time, τ. The determined τ values at each angle were plotted against the square of the scattering wave vector, q, to determine the apparent diffusion coefficient, D, according to the following equation: 4 −1 = 2 Apparent hydrodynamic radii (Rh) were then calculated using the Stokes-Einstein equation: where η is the solvent viscosity, kB is the Boltzmann constant and T is the absolute temperature.
Using SLS analysis at the same angles, partial Zimm plots were obtained and the radius of gyration (Rg) and the apparent aggregation number, Nagg, for each sample were calculated using the following equations: 4 where λ is the wavelength of the laser, nstandard is the refractive index of the standard, dn/dc is the refractive index increment of the sample and NA is Avogadro's number.
Small-Angle X-Ray Scattering. Small-angle X-ray scattering (SAXS) measurements were performed using

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Transmission Electron Microscopy Image Analysis. Dry-state TEM images were processed using ImageJ image analysis software to determine the population distribution of spherical and tubular polymersomes, the average maximum dimension (Save) (diameter for spherical polymersomes or length for tubesomes) of each sample, and the average tubesome length (Lave). Using ImageJ, the image threshold was first adjusted such that individual particles could be clearly resolved as dark shapes against a white background. The area (a) and perimeter (p) of each particle were then determined using the Analyze Particles feature, adjusting the area (a) and circularity (C) parameters such that a representative sample of particles was selected from each image. Image masks generated via this process are shown herein.
Next, circularity (C) values were calculated from the generated nano-object areas and perimeters using the equation shown in Figure S1. The circularity of each particle was exploited to differentiate its morphologyin general, particles with 1 ≥ C > 0.7 were assumed to be spherical polymersomes, whilst those with C ≤ 0.7 were assigned as cylindrical tubesomes. Based on this initial calculation, the fraction of cylindrical particles over spherical particles (FC) was determined, which was then benchmarked against the original dry-state TEM images by manually counting the number of spherical polymersomes (NS) and cylindrical tubesomes (NT) in a given area over multiple images. In some cases, the threshold value of C = 0.7 was adjusted such that the calculated cylindrical particle fraction matched the value obtained from manual counting.
The radius (r) for spherical polymersomes or length (l) for cylindrical tubesomes was then calculated from their areas using the corresponding equations shown in Figure S1 and averaged across the entire population to determine Dave and Lave, respectively. In all cases, a population of at least 300 particles were analyzed. For the P(NB-PEG)11-b-P(NB-MEG)260 sample, the tubesome fraction and Lave were determined by manually measuring the length of each particle over several images, as individual particles could not be isolated using the Analyze Particles feature of ImageJ software. Figure S1. Description of dry-state TEM image analysis procedure and equations utilized for calculation of nano-object circularity (C), radius (r) (for spherical polymersomes) and length (l) (for cylindrical tubesomes) values, using ImageJ image processing software.
Step-Growth Fitting of Image Analysis Data. For P(NB-PEG)11-b-P(NB-MEG)n diblock copolymer nano-objects, a process to generate step-growth fits relating the particle shape distribution to the average tubesome length was developed. For samples in which fusion was observed, the average number of spherical polymersomes that had fused per tubesome (NF) was calculated by dividing Lave by a fixed diameter, DC. This diameter was taken as the Dave of the sample prior to the onset of fusion, with the assumption that the width of the tubesomes remained constant across the series of targeted DPs once fusion began to occur. This assumption was supported by manual tubesome width measurements, which were found not to vary significantly over a large number of analyzed images. Carothers equations provide a relationship between monomer conversion, the degree of polymerization and the polymer dispersity for step-growth polymerizations. In our system, we treated the average number of polymersomes that had fused to make each tubesome (NF = Lave/Dave) and the fraction of cylindrical particles over spherical particles (FC) as analogies for the degree of polymerization and conversion, respectively. In this way, nonfused spherical polymersomes were considered to be "monomers", and tubesomes, comprised of two or more fused polymersomes, represented "polymers". Thus diblock copolymer nano-objects at 100-fold dilution were deposited on a glass slide and imaged by confocal laser scanning microscopy (CLSM) after evaporation of the solvent. Images were acquired using an Olympus FV3000 confocal laser Scanning Microscope. Samples were imaged at 60× magnification upon excitation using a 488 nm laser (green channel) and a 640 nm laser (red channel). Images were processed using cellSens (Olympus) and ImageJ image processing software.

Steady-State Fluorescence
Reduced Chi-square: Wk: Weighting factors for the individual data points; Sk: Measurement data points; Fk: Data points of the fitted curve; S10 N: The number of the free parameters which is approximately the number of fitted data points subtracted by the number of lifetime parameters used in the fit.
Intensity-average fluorescence lifetime (τAv,I) and amplitude-average fluorescence lifetime (τAv,A) values were determined from the fitting parameters according to the following equations:

Synthesis of exo-Norbornene imide Rhodamine B (NB-RhB)
The synthesis of exo-norbornene imide rhodamine B (NB-RhB) monomer was performed according to a previously described process with slight modification. 7 exo-Norbornene imide alcohol (205 mg, 1 mmol, 1 eq), rhodamine B (985 mg, 2 mmol, 2 eq) and DMAP (30.5 mg, 0.25 mmol. 0.25 eq) were dissolved in 10 mL of anhydrous DCM under a nitrogen atmosphere. A solution of DCC (412 mg, 2 mmol, 2 eq) in 5 mL of anhydrous DCM was added dropwise to the reaction mixture over a period of 30 min. The mixture was stirred for 24 h at room temperature, concentrated under reduced pressure, and purified via column chromatography using silica gel as the stationary phase and a mixture of CH2Cl2/MeOH 90:10 as the eluent to to afford the pure product of NB-RhB as a a purple solid (0.28 g, 45% S15 Figure S4. 1 H-NMR spectrum of exo-norbornene imide Rhodamine B (NB-RhB) in CDCl3. Figure S5. 13 C-NMR spectrum of exo-norbornene imide Rhodamine B (NB-RhB) in CDCl3.

Synthesis of P(NB-MEG)180 Homopolymer via Solution Ring-Opening Metathesis Polymerization (ROMP)
A typical procedure for the synthesis of P(NB-MEG)180 homopolymer via solution ROMP is described.

Supplementary Characterization Data for P(NB-amine)11-b-P(NB-MEG)n Diblock
Copolymer Nano-Objects Developed by Aqueous ROMPISA Figure S7. 1 H-NMR spectrum of P(NB-amine)11 macroinitiator in DMSO-d6.       Mn and ĐM values were calculated from PS standards using THF + 2% v/v NEt3 as the eluent. 83.2 ± 0.9 0.21 ± 0.01 84.7 ± 1.6 + 28.13 ± 2.35 ill-defined V + T a Dh and PD values measured from DLS analysis (the error shows the standard deviation from 4 repeat measurements). b Zeta potential values measured from microelectrophoretic analysis at pH = 2.0, using PB 2 (the error shows the standard deviation from at least 3 repeat measurements). c Morphologies observed from dry-state TEM imaging, using 1 wt% uranyl acetate (UA) solution for staining (Key: Wworm-like micelles, Vvesicles, Ttubesomes).   and 300) diblock copolymer nano-objects developed via aqueous ROMPISA and samples after aging for 4 weeks, stained with 1 wt% UA solution, along with summary of dry-state TEM image analysis results for aged P(NB-amine)11-b-P(NB-MEG)n diblock copolymer nano-objects, as determined by image processing using ImageJ analysis software.   1025 ± 90 0.41 ± 0.04 1157 ± 218 + 20.10 ± 0.50 T + L a Dh and PD values measured from DLS analysis (the error shows the standard deviation from 4 repeat measurements). b Zeta potential values measured from microelectrophoretic analysis at pH = 2.0, using PB 2 + 100 mM NaCl (the error shows the standard deviation from at least 3 repeat measurements). c Morphologies observed from dry-state TEM imaging, using 1 wt% uranyl acetate (UA) solution for staining (Key: Vvesicles, Ttubesomes, Llamellae).

Supplementary Characterization Data for P(NB-PEG)11-b-P(NB-MEG)n Diblock
Copolymer Nano-Objects Developed by Aqueous ROMPISA Figure S25. 1 H-NMR spectrum of P(NB-PEG)11 macroinitiator in DMSO-d6.       Mn and ĐM values were calculated from PS standards using THF + 2% v/v NEt3 as the eluent. Macroscopic precipitation -Unstable particles a Dh and PD values measured from DLS analysis (the error shows the standard deviation from 4 repeat measurements). b Zeta potential values measured from microelectrophoretic analysis at pH = 2.0, using PB 2 (the error shows the standard deviation from at least 3 repeat measurements). c Morphologies observed from dry-state TEM imaging, using 1 wt% uranyl acetate (UA) solution for staining (Key: Vvesicles, Ttubesomes).           Figure S46. 1 Table S15. Summary of Dh and PD values and observed morphologies for P(NB-PEG)11-b-P(NB-

Sample
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