Triggered Polymersome Fusion

The contents of biological cells are retained within compartments formed of phospholipid membranes. The movement of material within and between cells is often mediated by the fusion of phospholipid membranes, which allows mixing of contents or excretion of material into the surrounding environment. Biological membrane fusion is a highly regulated process that is catalyzed by proteins and often triggered by cellular signaling. In contrast, the controlled fusion of polymer-based membranes is largely unexplored, despite the potential application of this process in nanomedicine, smart materials, and reagent trafficking. Here, we demonstrate triggered polymersome fusion. Out-of-equilibrium polymersomes were formed by ring-opening metathesis polymerization-induced self-assembly and persist until a specific chemical signal (pH change) triggers their fusion. Characterization of polymersomes was performed by a variety of techniques, including dynamic light scattering, dry-state/cryogenic-transmission electron microscopy, and small-angle X-ray scattering (SAXS). The fusion process was followed by time-resolved SAXS analysis. Developing elementary methods of communication between polymersomes, such as fusion, will prove essential for emulating life-like behaviors in synthetic nanotechnology.

1. General information and abbreviations 1

.1 Materials
Unless stated otherwise, reagents were obtained from commercial sources and used without purification. Tetrahydrofuran (THF) (HPLC grade) was purchased from VWR Chemicals and was purified via passage through a column of neutral alumina prior to use. Formvar-carbon coated (300 mesh) and lacey-carbon coated (400 mesh) copper grids were purchased from EM Resolutions.
Flash column chromatography was carried out using silica (particle size 40-63 μm) as the stationary phase. TLC was performed on precoated silica gel plates and visualized using short wave ultraviolet light in combination with standard laboratory stains (basic potassium permanganate and iodine vapour).

Characterization Techniques
NMR Spectroscopy. 1 H NMR and 13 C NMR spectra were recorded at 300 MHz or 400 MHz on a Bruker DPX-300 or a Bruker DPX-400 spectrometer in CDCl3, (CD3)2SO or D2O. Chemical shifts of protons are reported as δ in parts per million (ppm) and are relative to solvent residual peaks (CDCl3 δ = 7.26 ppm, (CD3)2SO δ = 2.50 ppm, D2O δ = 4.79 ppm). All 1 H resonances are reported to the nearest 0.01 ppm. The multiplicity of 1 H signals are indicated as: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; or combinations of thereof. Coupling constants (J) are quoted in Hz and reported to the nearest 0.1 Hz. Where appropriate, averages of the signals from peaks displaying multiplicity were used to calculate the value of the coupling constant. 13 C NMR spectra were recorded on the same spectrometer with the central resonance of the solvent peak as the internal reference (CDCl3 δ = 77.16 ppm). All 13 C resonances are reported to the nearest 0.01 ppm.
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.
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. Samples were diluted with PB2 to give a final polymer concentration of 0.01 wt%. 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 four times S4 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 anisotropic particles DLS was used to indicate a change in average particle size and obtain dispersity information.
Transmission Electron Microscopy. Dry-state stained transmission electron microscopy (TEM) imaging was performed on a JEOL JEM-1400 microscope operating at an acceleration voltage of 80 kV. All dry-state samples were diluted with PB2 to an appropriate analysis concentration and then deposited onto formvar-coated grids. After roughly 1 min, excess sample was blotted from the grid and the grid was then stained with an aqueous 1 wt% uranyl acetate (UA) solution for 1 min prior to blotting, drying and microscopic analysis. Cryogenic transmission electron microscopy (cryo-TEM) imaging was performed on a JEOL JEM-2100Plus microscope operating at an acceleration voltage of 200 kV. Samples for cryo-TEM analysis were prepared, after dilution with PB2, by depositing 8 μL of sample onto a lacey carbon grid followed by blotting for approximately five seconds. The grid was then plunged into a pool of liquid ethane, cooled using liquid nitrogen, to vitrify the sample. Transfer into a pre-cooled cryo-TEM holder was performed under liquid nitrogen temperatures prior to microscopic analysis. Images were analyzed using ImageJ.
Differential Scanning Calorimetry. Determination of the glass transition temperatures (Tg) was performed using a Mettler Toledo DSC 3 differential scanning calorimeter by heating the sample from 25 °C to 200 °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.
Small-angle X-ray scattering. SAXS patterns were recorded at a synchrotron source (Diamond Light Source, station I22, Didcot, UK; Experiment ID SM28511) using monochromatic X-ray radiation (X-ray wavelength λ = 1.00 Å, with scattering vector q ranging from 0.0017 to 0.17 Å -1 , where q = 4π sin θ/λ and θ is one-half of the scattering angle) and a 2D Pilatus 2M pixel detector (Dectris, Switzerland). All static SAXS measurements were performed on 1.0% w/w copolymer dispersions in 2.0 mm glass capillaries. Time-resolved experiments were performed using a BioLogic SFM-400 stopped-flow mixing system equipped with an umbilical connector, mixing unit and observation cell containing a 1.0 mm glass capillary, without delay lines: a 1.0% w/w polymersome dispersion in pH2 water and aqueous NaOH were loaded into two separate 10 mL syringes before 200 μL of each solution was flowed into the observation cell at 1.0 mL s −1 so that a final solution pH of 12 and a copolymer concentration of 0.5% w/w was obtained on mixing. Scattering data were reduced and normalized, with glassy carbon being used for the absolute intensity calibration, utilizing standard routines available at the beamline 3 and further analyzed (background subtraction and data modelling) using Irena SAS macros for Igor Pro. 4 S5 2. Evaluation of P(NB-amine) 5 hydrophobicity LogPoct Analysis. Octanol-water partition coefficients (LogPoct) for P(NB-amine)5 were calculated in Materials Studio 2020, 5 using an atom-based approach (ALogP98 method) 6 for a molecular model containing C, H, N, and O atoms.
Surface Area Analysis. The octanol-water partition coefficient (LogPoct) was normalized by solvent accessible surface area (SA) using Materials Studio 2020. First, the oligomer was subjected to a Geometry Optimization procedure using the Forcite Molecular Dynamics (MD) module with a COMPASS II force field. The force field contains information on important parameters, like preferred bond lengths, bond angles, torsion angles, partial charges, and van der Waals radii that influence the conformation. 7 To minimize energy and determine a preferred conformation, this simulation ran until the energy of the oligomer decreased below predetermined convergence criteria (1 × 10 -4 kcal mol -1 energy convergence, 0.005 kcal mol -1 /Å force convergence, and 5 × 10 -5 Å displacement convergence). Second, the SA value represents the Connolly surface area created by an algorithm that rolls a ball over the surface of the oligomer. To ensure the SA values are meaningful in the context of octanolwater partition coefficients (LogPoct), the probe had a 1.4 Å radius to match the size of a water molecule. Third, the oligomer was annealed for 200 cycles using a sinusoidal temperature ramp (300 -700 K) to maximize variability in SA values.

Figure S1
Model used for hydrophobicity analysis.

Synthesis of monomers
3.1 Synthesis of NB-PEG via S1 NB-PEG Scheme S1 Synthesis of NB-PEG from S1.
Two aliquots (50 μL, 0.22 μmol, for P1200 and 33 μL, 0.15 μmol, for P1300) of the resulting solution of P(NB-PEG)11-b-P(NB-amine)5 in THF were dispensed into 2 mL glass vials containing a stirrer bar. Filtered THF was added to give 100 μL in total in each vial. A solution of NB-MEG (10 mg, 45 μmol, 200 eq. for P1200 and 300 eq. for P1300) in 0.9 mL of acidic phosphate buffer (pH = 2, PB2, final solids concentration = 1 wt%) was added rapidly to each vial. The resulting solution was thoroughly mixed by drawing up the entire volume into the pipette tip and ejecting the liquid back into the vial three times. The ROMPISA polymerizations were stirred at 300 rpm for 30 minutes to give P(NB-PEG)11-b-P(NB-amine)5-b-P(NB-MEG)200/300 (P1200 and P1300 respectively). These were analyzed by 1 H NMR, GPC, DLS, TEM and SAXS (for P1200 only, section 10). DSC was performed on freeze dried P1200 (section 6).
S19 Figure S8 Cryo-TEM images of P1300 particles as synthesized at pH 2. 11 -r-P(NB-amine) 5  Two aliquots (50 μL, 0.22 μmol, for P2200 and 33 μL, 0.15 μmol, for P2300) of the resulting solution of P(NB-PEG)11-r-P(NB-amine)5 in THF were dispensed into 2 mL glass vials containing a stirrer bar. Filtered THF was added to give 100 μL in total in each vial. A solution of NB-MEG (10 mg, 45 μmol, 200 eq. for P2200 and 300 eq. for P2300) in 0.9 mL of acidic phosphate buffer (pH = 2, PB2, final solids concentration = 1 wt%) was added rapidly to each vial. The resulting solution was thoroughly mixed by drawing up the entire volume into the pipette tip and ejecting the liquid back into the vial three times. The ROMPISA polymerizations were stirred at 300 rpm for 30 minutes to give P(NB-PEG)11-r-P(NB-amine)5-b-P(NB-MEG)200/300 (P2200 and P2300 respectively). These were analyzed by 1 H NMR, GPC, DLS and TEM. DSC was performed on freeze dried P2200 (section 6).   Cryo-TEM Figure S13 Cryo-TEM images of P2200 particles as synthesized at pH 2.

Triggered fusion of P1 200 and P2 200
To 100 μL solution of P1200 or P2200 (as synthesized, 10 vol% THF in PB2) was rapidly added 300 μL of NaOH solution (100 mM, 10 vol% THF). The resulting solution was thoroughly mixed by drawing up the entire volume into the pipette tip and ejecting the liquid back into the vial three times. The resulting solution was analyzed by GPC, DLS, TEM and SAXS (for P1200 only, see section 10).  Dry-state TEM Figure S16 Dry-state TEM image and histogram (300 particles analyzed) of P1200 after triggered fusion.

Characterization Summary
Figure S17 Dry-state TEM images of P2200 particles after triggered fusion.

Figure S19
Cryo-TEM images of P2200 particles after triggered fusion. Figure S20 DSC thermogram of P1200 and P2200

Reacidification of fused P1200
To 100 μL solution of fused P1200 (0.25 wt%, fusion triggered as above) at pH 12 was added 300 μL of PB2 solution (100 mM, 10 vol% THF) to readjust the pH to 2. After five minutes the resulting solution was analyzed by DLS and TEM.

S32
Dry-state TEM Figure S25 Dry-state TEM image and histogram (300 particles analyzed) of P3200 as synthesised at pH 2.

Attempted triggered fusion of P3200
To 100 μL solution of P3200 (as synthesized, 10 vol% THF in PB2) was rapidly added 300 μL of NaOH solution (100 mM, 10 vol% THF). The resulting solution was thoroughly mixed by drawing up the entire volume into the pipette tip and ejecting the liquid back into the vial three times. The resulting solution was analyzed by DLS and TEM. Figure S26 DLS traces of P3200 particles as synthesized after attempted triggered fusion.

DLS
. S33 Figure S27 Dry-state TEM image and histogram (300 particles analyzed) of P3200 after attempted triggered fusion.

Interrupted fusion experiments
Fusion of P1200 particles was triggered as detailed in section 6. 40 μL aliquots were taken at five seconds and five minutes and immediately quenched in 960 μL PB2 solution (i.e. diluted to the concentration used for DLS analysis and TEM grid preparation) and analyzed.  A solution of NB-PEG (31.2 mg, 59 μmol, 13 eq.) in 530 μL of filtered THF was rapidly added to a solution of G3 (3.8 mg, 5.2 μmol, 1.2 eq.) in 120 μL of THF contained within a glass vial equipped with a stirrer bar. The resulting solution was stirred rapidly for five minutes to give P(NB-PEG)11. After this time a 100 μL aliquot was removed for GPC analysis, leaving 1.0 eq. of P(NB-PEG)11 in 550 μL of THF.

Characterization Summary
To this solution was added NB-amine (2.6 mg, 11 μmol, 2.5 eq.) in 450 μL THF. The resulting solution was stirred for a further five minutes to give P(NB-PEG)11-b-P(NB-amine)2.5. After this time a 100 μL aliquot was removed for GPC analysis, leaving 0.9 eq. of P (

S39
Dry State TEM Figure S33 Dry-state TEM image and histogram (300 particles analyzed) of P4200 particles as synthesized at pH 2.

Triggered fusion of P(NB-PEG)11-b-P(NB-amine)2.5-b-P(NB-py)2.5-b-P(NB-MEG)200, P4200
To 100 μL solution of P4200 (as synthesized, 10 vol% THF in PB2) was rapidly added 100 μL of NaOH solution (100 mM, 10 vol% THF in deionised water) to give a pH 7 solution. The resulting solution was thoroughly mixed by drawing up the entire volume into the pipette tip and ejecting the liquid back into the vial three times. A 20 μL aliquot was taken for analysis by TEM and DLS. A further 200 μL of NaOH solution was added to adjust the remaining solution to pH 12. The resulting solution was analyzed by DLS and TEM.  Figure S34 DLS traces of P4200 particles after triggered fusion to pH 7 and pH 12.

S41
Dry-state TEM Figure S35 Dry-state TEM image and histogram (300 particles analysed) of P4200 particles after pH 7 trigger.

Figure S36
Dry-state TEM image and histogram (300 particles analysed) of P4200 particles after pH 12 trigger.

SAXS Modelling
Programming tools within the Irena SAS macros for Igor Pro were used to model experimental SAXS data. 7 Models used to fit SAXS data in this work were previously developed by Pedersen et al. 8 In general, the intensity of X-rays scattered by a dispersion of nano-objects [as represented by the scattering cross-section per unit sample volume, (q)] can be expressed as: where ( , , … , ) is the form factor, , … , is a set of k parameters describing the nano-object structural morphology, Ψ , … , is the distribution function, S(q) is the structure factor and N is the number density of nano-objects per unit volume expressed as: where ( , … , ) is the nano-object volume and is the volume fraction of the nano-objects within the dispersion. It is assumed that S(q) = 1 at the sufficiently low copolymer concentrations used in this study (≤1.0% w/w).
For this study, experimental SAXS data required fitting to either the spherical micelle model (e.g. static data obtained at pH2), cylindrical micelle (e.g. static data obtained at pH12), or a combination of the two (e.g. all time-resolved data). Additionally, optimum fits often required the use a power law relationship (herein described as a unified fit) and/or a low-intensity background to adequately fit lowq and high-q data, respectively. Thus, the intensity of scattering at a given q vector, ( ), is expressed as: where ( ) is the form factor for spherical micelles, ( ) is the form factor for cylindrical micelles, and terms , and are constants.

Spherical micelle model
The spherical micelle form factor for Equation S1, which contributes to ( ) , is given by: where is the volume-average sphere core radius and is the radius of gyration of the coronal steric stabilizer block (in this case, P(NB-PEG), which was fixed at 32.6 Å for data fits). The X-ray scattering length contrasts for the core and corona blocks are given by = ( − ) and = ( − ) respectively. Here, , and are the X-ray scattering length densities of the core block ( ( ) = 10.77 x 10 10 cm -2 ), corona block ( ( ) = 10.31 x 10 10 cm -2 ) and water ( = 9.42 x 10 10 cm -2 ), respectively. and are the volumes of the core block ( ( ) = 61750 Å 3 ) and the corona block ( ( ) = 8600 Å 3 ), respectively. Values for and were calculated using = taking the solid-state homopolymer densities of P(NB-MEG) determined by helium pycnometry ( ( ) = 1.19 g cm -3 ) and P(NB-PEG), which was taken to equal that of PEG ( ( ) = 1.13 g cm -3 ), 9 where is the number-average molecular weight of each polymer block determined by 1 H NMR spectroscopy. The sphere form factor amplitude is used for the amplitude of the core self-term: . A sigmoidal interface between the two blocks was assumed for the spherical micelle form factor (Equation S4). This is described by the exponent term with a width accounting for a decaying scattering length density at the micellar interface. This value was fixed at 2.2 during fitting.
The form factor amplitude of the spherical micelle corona is: The radial profile, ( ), can be expressed by a linear combination of two cubic b splines, with two fitting parameters and corresponding to the width of the profile and the weight coefficient respectively. This information can be found elsewhere, 10,11 as can the approximate integrated form of Equation S5. The self-correlation term for the coronal block is given by the Debye function: where is the radius of gyration of the P(NB-PEG) coronal block. The aggregation number, , of the spherical micelle is given by: where is the volume fraction of solvent within the P(NB-MEG) micelle cores, which was found to be zero in all cases. A polydispersity for one parameter ( ) is assumed for the micelle model, which is described by a Gaussian distribution. Thus, the polydispersity function in Equation S1 can be represented as: where is the standard deviation for . In accordance with Equation S2, the number density per unit volume for the micelle model is expressed as: where is the total volume fraction of copolymer in the spherical micelles and ( ) is the total volume of copolymer within a spherical micelle [ ( ) = ( + ) ( )].

Cylindrical micelle model
The cylindrical micelle form factor for Equation S1 is given by: where all the parameters are the same as those described in the spherical micelle model (Equation S4), unless stated otherwise.

S45
The self-correlation term for the cylinder core cross-sectional volume-average radius is: and is the first-order Bessel function of the first kind, and a form factor ( , , ) for selfavoiding semi-flexible chains represents the cylindrical micelles, where is the Kuhn length and is the mean contour length. In all cases when applying the cylindrical micelle model, was found to equal the value of , indicating the presence of rigid cylinders. A complete expression for the chain form factor can be found elsewhere. 12 The mean aggregation number of the cylindrical micelle, , is given by: Again, was found to be zero in all cases. The possible presence of semi-spherical caps at both ends of each worm is neglected in this form factor.
A polydispersity for one parameter ( ) is assumed for the cylindrical micelle model, which is described by a Gaussian distribution. Thus, the polydispersity function in Equation S1 can be represented as: where is the standard deviation for . In accordance with Equation S2, the number density per unit volume for the worm-like micelle model is expressed as: where is the total volume fraction of copolymer in the cylindrical micelles and ( ) is the total volume of copolymer in a cylindrical micelle ( ) = ( + ) ( ) .  Figure S37 SAXS data of P1200 particles as synthesized at pH 2 (red line) and one second after mixing in stopped-flow capillary (black line).