Preparation of Membrane Protein–Functionalized Polymer and Polymer/Lipid Hybrid Large and Giant Unilamellar Vesicles

Summary

This protocol demonstrates detergent-mediated reconstitution of a membrane protein, cytochrome bo3 ubiquinol oxidase, into large unilamellar vesicles made of PDMS-g-PEO and their conversion into giant vesicles via fusion–electroformation. The resulting functional polymer-based compartments are suitable for advanced biochemical and biophysical studies.

Abstract

Vesicles formed from amphiphilic copolymers, alone or blended with phospholipids, offer superior mechanical and chemical stability compared to conventional lipid vesicles. This makes them an attractive chassis, one that can be further developed and expanded to enable specific applications, such as drug delivery, diagnostic biosensing, construction of artificial cells, and bioinspired micro- and nanoreactors. In particular, functionalization through membrane protein incorporation is essential for many of these applications. Here, we present a protocol for preparing membrane protein–functionalized large unilamellar vesicles (LUVs) from the graft copolymer PDMS-g-PEO via detergent-mediated reconstitution. Furthermore, we describe how these large vesicles can be converted to giant unilamellar vesicles (GUVs) using a fusion–electroformation approach. The protocol covers fluorescence labeling of membrane proteins, preparation of polymer and hybrid LUVs, membrane protein reconstitution, size distribution analysis via dynamic light scattering (DLS), assessment of protein activity via oxygen consumption measurements, preparation of protein-functionalized GUVs, and analysis of protein insertion and proton pumping activity in GUVs via confocal microscopy. Representative results demonstrate the formation of monodisperse proteo-LUVs with a polydispersity index (PDI) below 0.2, and successful generation of proteo-GUVs ranging from 5–35 µm in diameter. Protein activity is confirmed by oxygen consumption measurements in both polymer LUVs (18.2 nmol/min/mL) and hybrid LUVs (26.9 nmol/min/mL). Protein insertion into GUVs is quantified via fluorescence intensity, yielding 20.6 ± 3.7 a.u. for polymer GUVs and 26.2 ± 5.0 a.u. for hybrid GUVs. Proton pumping activity in GUVs, monitored via an encapsulated pH-sensitive dye, is consistent with protein functionality, with inward proton pumping being predominant. The copolymer’s mechanical softness and lipid-like bilayer thickness (~5.3 nm) support efficient protein insertion and preservation of functionality.

Introduction

Amphiphilic block copolymers can self-assemble in aqueous solution into membranes, which spontaneously curve and close upon themselves to form spherical, membrane-enclosed compartments known as polymersomes (polymer vesicles). Compared to lipid membranes, polymer-based compartments offer increased chemical versatility and enhanced chemical¹ and mechanical stability², making them highly attractive for applications such as drug delivery, nano- and microreactors, and the construction of durable artificial cells. Both our group and others have functionalized polymer membranes by inserting membrane proteins to create artificial organelles and cells^{1,3,4,5,6,7,8,9,10,11,12,13,14,15}. Polymers containing a poly(dimethylsiloxane) (PDMS) hydrophobic core are particularly well suited for membrane protein incorporation due to the high chain flexibility and the possibility to tune the membrane hydrophobic thickness to better match the protein’s transmembrane domain⁵. While our laboratory has primarily concentrated on the graft copolymer poly(dimethylsiloxane)-graft-poly(ethylene oxide) (PDMS-g-PEO), which forms highly dynamic membranes^4,16,17 that are softer than conventional lipid bilayers and exhibit a thickness of approximately 5.3 nm¹, other groups have successfully employed PDMS-based triblock copolymers such as PMOXA-b-PDMS-b-PMOXA^{5,6,7,8,9,10,11} and PEtOz-b-PDMS-b-PEtOz¹² for membrane protein reconstitution. Even comparatively rigid diblock copolymers such as PBD-b-PEO, particularly in hybrid systems mixed with lipids, have been utilized for successful protein incorporation¹³. These examples highlight the versatility of polymer-based membranes as reconstitution platforms, which can additionally provide enhanced chemical stability¹ and prolonged functional lifetime¹⁴ for incorporated membrane proteins. Although polymer/lipid hybrid membranes may not always match the chemical stability of pure polymer systems¹, they offer an attractive compromise by combining the mechanical robustness of polymers with the biological relevance of lipids. This results in a membrane environment that more closely mimics native biological membranes while preserving key advantages of synthetic materials. Such hybrid architectures can offer additional advantages, including reduced proton permeability arising from nanoscale rearrangement¹ and domain formation within the membrane¹⁵.

Several techniques exist for reconstituting membrane proteins into nanosized vesicles (LUVs), including organic solvent-mediated reconstitution (e.g., reverse-phase evaporation, rehydration of lipid-protein films), mechanical approaches (e.g., sonication, French press, freeze-thaw), and detergent-mediated reconstitution (via detergent removal, dilution, or direct incorporation)¹⁸, and more recent strategies such as direct transfer from styrene-maleic acid copolymer nanodiscs¹⁹. While porins have been successfully incorporated into polymer LUVs through direct insertion, for example, by adding the protein to the polymer film during rehydration⁸. This approach generally results in low insertion efficiency for larger or more complex membrane proteins and often leads to random protein orientation. Consequently, detergent-mediated reconstitution, which involves controlled addition of detergent followed by its removal, remains the most widely used method. It is also the preferred strategy for incorporating larger membrane proteins into polymer-based LUVs, particularly for asymmetric proteins where controlled orientation is crucial for function. In this approach, membranes of vesicles are first saturated with the detergent of choice (saturating detergent concentration is denoted as R_sat), partially solubilized (coexistence of vesicles and micelles), or fully solubilized, i.e., broken down into micelles (detergent concentration is denoted as R_sol). This initial membrane treatment is then followed by protein addition, incubation, and detergent removal¹⁸. The latter can be achieved via dialysis, size-exclusion chromatography, or adsorption onto hydrophobic polystyrene beads (e.g., Bio-Beads). The choice of detergent, its concentration, and the removal method are optimized for each membrane type and protein to maximize incorporation efficiency and control protein orientation²⁰.

While LUVs are well-suited for bulk activity measurements, single-vesicle studies and optical microscopy–based analyses require membrane proteins to be reconstituted into giant unilamellar vesicles (GUVs). Five main approaches are used for protein incorporation into GUVs: co-formation in the presence of protein and organic solvent²¹, co-formation in the presence of a detergent²², insertion into preformed GUVs via detergent dilution²³, insertion via membrane fusion with proteo-LUVs^16,24, and fusion-electroformation²⁵. While these approaches have been successfully applied to lipid GUVs, their transfer to polymer or polymer/lipid hybrid GUVs is often challenging. In particular, co-formation and detergent-dilution methods frequently result in low insertion efficiencies in polymer membranes or are incompatible with certain polymer compositions¹⁵. Fusion-based strategies^{4,16,24,26,27} that rely on additional fusogenic agents may also be problematic, as synthetic cationic lipids/polymers or fusogenic peptides can compromise membrane protein stability, alter membrane properties, or reduce the incorporation efficiency of the target protein. Fusion-electroformation overcomes many of these limitations and has proven to be a robust and efficient method for incorporating membrane proteins into polymer and polymer/lipid hybrid GUVs^1,5,15. This approach enables controlled protein incorporation while preserving membrane integrity and protein functionality, making it particularly suitable for the generation of protein-functionalized polymer GUVs.

Reconstituting proton pumps into polymer-based vesicles is essential for constructing durable artificial cells, as it enables the generation of transmembrane proton gradients—the fundamental energy currency of living systems. By recreating chemiosmotic energy conversion in a controlled minimal membrane environment²⁸, these vesicles can drive ATP synthesis, active transport, and metabolic reactions, establishing the energetic foundation required for autonomous, life-like functionality. One representative proton pump used in such systems is cytochrome bo₃ oxidase from E. coli, a terminal respiratory enzyme that couples oxygen reduction to proton translocation across the membrane. Because electron transfer and proton pumping are tightly coupled, their activity in synthetic vesicles can be quantitatively monitored either by oxygen consumption^1,20 or by tracking proton gradient formation¹.

In this protocol, we present a detailed workflow for the preparation of PDMS-g-PEO-based LUVs, including pure polymer and polymer/lipid hybrid membranes, their functionalization with membrane proteins, scale-up to GUVs, and activity measurements of the reconstituted proton pump cytochrome bo₃ oxidase through both bulk oxygen consumption assays and single-vesicle proton pumping measurements.

Protocol

NOTE: All hazardous chemical waste generated during this protocol must be disposed of in accordance with institutional and local regulations.

1. Preparation for Bio-Beads SM-2

NOTE: Bio-Beads SM-2 are prewashed with methanol to remove preservatives and organic contaminants prior to detergent adsorption.

1. Place filter paper in a glass funnel positioned over a clean glass beaker or flask.
2. Add at least 600 mg of Bio-Beads SM-2 to the funnel (sufficient for 3 repeats of reconstitution for both hybrid and polymer LUVs).
   NOTE: A minimum of 90 mg of Bio-Beads SM-2 is required per reconstitution, as specified in step 4.4.1. A larger amount can be prepared if more reconstitutions are planned.
3. Wash the beads three times with pure methanol, using sufficient volume to fully immerse the beads each time.
   CAUTION: Perform all methanol handling steps in a chemical fume hood.
4. Immediately wash the beads three times with Milli-Q water to remove residual methanol.
   NOTE: Do not allow the beads to run dry during the washing procedure. When removing wash solutions, ensure the beads remain damp and are not allowed to become completely dry.
5. Transfer the washed beads to a suitable storage container (e.g., a sterile 50 mL polypropylene centrifuge tube or glass bottle with a tightly sealed cap), add Milli-Q water until the beads are completely submerged, and store at 4 °C for up to 3 months.

2. Fluorescence labeling of membrane proteins

1. Protein dilution
   1. Dissolve cytochrome bo₃ oxidase to 2.2 mg/mL in 20 mM HEPES buffer containing 0.05% n-dodecyl β-D-maltoside (DDM) (w/v), pH 8.3. This pH ensures a sufficient fraction of unprotonated (reactive) primary amines for efficient labeling.
      ​NOTE: For optimal labeling with ATTO N-hydroxysuccinimidyl (NHS) dyes, maintain the buffer pH between 8.3 and 8.5. For other dyes, follow the manufacturer’s instructions for protein conjugation, including the recommended labeling conditions.
2. Dye addition
   1. Add an eightfold molar excess of amine-reactive ATTO 643 N-hydroxysuccinimidyl (NHS) ester, dissolved at 2 mg/mL in anhydrous DMSO, to the protein solution.
      ​NOTE: For other dyes, follow the manufacturer’s instructions for protein conjugation, including the recommended dye-to-protein molar excess.
   2. Mix gently by tapping the microcentrifuge tube three times to ensure homogeneous distribution of the dye.
3. Incubation
   1. Incubate the reaction mixture at room temperature (20–25 °C) for 1.5 h under gentle agitation (250 rpm) in a thermomixer, in the dark to prevent photobleaching.
4. Removal of unbound dye
   1. Equilibrate the Superdex 200 10/300 GL column (CV: 23.562 mL) with at least 2 column volumes (approximately 48 mL) of membrane protein elution buffer (20 mM HEPES, pH 7.5, 200 mM KCl, 0.05% DDM (w/v)) at a flow rate of 0.3 mL/min using an ÄKTA purification system.
      NOTE: For removal of excess fluorescent dye, shorter gel filtration columns (10–20 cm bed length) are generally sufficient, particularly for less hydrophilic dyes. The dye manufacturer recommends desalting using a Sephadex G-25 matrix. A Superdex 200 10/300 GL column was used in this study due to the availability of the instrument.
   2. Load up to 500 µL of the labeled protein using a 1 mL capillary loop and elute with the same buffer at 0.3 mL/min.
   3. Monitor elution by measuring absorbance at 280 nm for protein and 643 nm for ATTO 643 dye.
   4. Collect 0.5 mL fractions over 1.5 column volumes. Identify fractions containing both protein and dye. Use directly if sufficiently concentrated, or concentrate using Amicon centrifugal filter units. For concentration using Amicon filters, use a centrifugal filter with a 30 kDa molecular weight cutoff and centrifuge at 14,000 × g for 30 min at 4 °C.
5. Determination of protein concentration and degree of labeling (DOL)
   1. Measure absorbance spectra of the purified conjugate.
   2. Calculate protein concentration and the average number of dye molecules per enzyme using:
      
      where A_max represents the absorbance of the conjugate at the dye absorption maximum (λ_abs; ATTO 643, λ = 643 nm), A₂₈₀ is the absorbance at 280 nm (absorption maximum of proteins), ε_prot is the molar extinction coefficient of denatured cytochrome bo₃ oxidase (184,720 M⁻¹cm⁻¹), ε_dye is the molar extinction coefficient of the dye (150,000 M⁻¹cm⁻¹), and CF₂₈₀ is correction factor for ATTO 643 absorbance at 280 nm (0.04).

3. Preparation of large unilamellar vesicles (LUVs)

1. Preparation of thin polymer or polymer/lipid film
   1. Use positive displacement pipettes with solvent-resistant tips or gas-tight glass syringes to transfer polymer, lipid, and fluorescent dye solutions dissolved in chloroform:methanol (2:1, v/v) into a clean glass vial according to the compositions listed in Table 1.
      CAUTION: Conduct all solvent handling under a fume hood while wearing protective gloves and safety glasses.
   2. Mix the solution thoroughly by gentle vortexing while holding the vial upright to ensure homogeneity.
      CAUTION: Adjust the vortex speed carefully to prevent the liquid from splashing or squirting out of the vial onto your hands.
   3. Evaporate the organic solvents under a gentle stream of nitrogen in the fume hood for approximately 1 h, until a uniform thin film forms on the vial wall (Figure 1).
      NOTE: Adjust the nitrogen flow so the liquid moves gently without splashing; too high a flow can cause uneven film formation. After 5–10 min, the flow may be slightly increased to ensure complete drying and even spreading of the vesicle mixture along the vial bottom.
2. Film rehydration
   1. Rehydrate the dried film with 1 mL of rehydration buffer (1 mM Tris-HCl, pH 7.5, 200 mM sucrose) to obtain a final amphiphile concentration of 5 mg/mL (corresponding to 1.67 mM for polymersomes and 2.14 mM for hybrid vesicles). If polymersomes are used for reconstitution, supplement the rehydration buffer with 0.1% sodium cholate.
      ​CAUTION: Sodium cholate is a mild irritant. Avoid prolonged skin contact and inhalation of powder. Handle according to the manufacturer's safety data sheet (SDS).
   2. Vortex vigorously until the film is completely detached and suspended, forming multilamellar vesicles (MLVs).
3. Freeze–thaw cycles (hybrid vesicles only)
   1. Subject hybrid MLV suspensions to five freeze–thaw cycles as follows: freeze the samples in liquid nitrogen for 1 min, thaw them in a 35 °C water bath until completely melted, and vortex at 2500–3000 rpm for 30 s using a tube shaker. Repeat this procedure for a total of five cycles.
      NOTE: Skip this step for pure polymersomes.
      CAUTION: Liquid nitrogen (−196 °C) can cause severe cryogenic burns. Handle in a well-ventilated area wearing appropriate PPE (cryogenic gloves, safety goggles, and a lab coat). Avoid contact with skin and eyes.
4. Extrusion
   1. Transfer the vesicle suspension to a mini-extruder equipped with a 100 nm pore size polycarbonate membrane.
   2. Extrude the suspension 21 times to obtain size-homogeneous LUVs.
      ​NOTE: Ensure that the final extrusion step leaves the LUV suspension in the syringe opposite to the initial loading syringe. This minimizes contamination of the final sample with larger vesicles or residual material that may not have fully passed through the membrane.
5. Storage
   1. Store the LUV suspension at 4 °C and use it within one week to maintain vesicle size distribution and prevent aggregation or microbial growth in the sucrose-containing buffer.
   2. Before use, gently homogenize the LUV suspension by vortexing.

4. Reconstitution of membrane protein into LUVs

1. Pretreatment of vesicles with detergent
   1. Transfer 200 µL of LUVs into a 2 mL microcentrifuge tube (Figure 1). If using hybrid LUVs, add 1 µL of 20% (w/v) sodium cholate to obtain a final detergent concentration of 0.1% (w/v). If using polymer LUVs, no additional detergent is needed as sodium cholate is already present.
      ​NOTE: Prepare the detergent stock in the same buffer used for the experiments to ensure compatibility with the vesicles.
   2. Gently mix by lightly tapping the microcentrifuge tube three times. Avoid vigorous agitation or vortexing, which can create foam and introduce air bubbles.
2. Introduction of protein to detergent-treated vesicles
   1. Add cytochrome bo₃ oxidase (for activity measurements) or cytochrome bo₃ oxidase–ATTO 643 (for fluorescence imaging) to the detergent-destabilized vesicles to achieve the following final protein concentrations: for polymersomes, 0.556 µM for LUV experiments and 1.112 µM for GUV experiments; for hybrid vesicles, 0.714 µM for LUV experiments and 1.428 µM for GUV experiments.
      NOTE: These concentrations correspond to a protein-to-polymer molar ratio of 1:3,000 for LUVs and 1:1,500 for GUVs.
   2. Add the protein slowly while gently mixing, with the goal of distributing it evenly throughout the solution and avoiding local overconcentration.
      NOTE: The volume of protein added may vary between fractions due to differences in protein stock concentration.
3. Assembly of protein–detergent–polymer complexes
   1. Incubate the mixture at 4 °C for 30 min with mild agitation (400 rpm) in a thermomixer to allow formation of mixed protein–detergent–polymer assemblies prior to detergent removal.
4. Detergent removal
   1. Remove detergent by sequential addition of Bio-Beads SM-2 (Bio-Rad). Remove 30 mg of Bio-Beads from the storage solution, ensuring they remain damp (wet but without excess liquid). Add the beads to the sample and incubate for 30 min at 4 °C with gentle agitation at 600 rpm in a thermomixer. Repeat this step two additional times, for a total of 90 mg Bio-Beads and a total incubation time of 1.5 h.
      NOTE: Use only fully hydrated bio-beads. Do not transfer dry beads, beads adhering to the walls of the Bio-Beads stock tube, or beads floating at the surface. If beads adhere to the wall of the reconstitution reaction tube above the liquid level after addition, briefly centrifuge the tube (e.g., 3–5 s in a benchtop minicentrifuge) to collect the beads at the bottom before incubation.
   2. Pellet the Bio-Beads by centrifugation at 2,000 × g for 2 min using a benchtop mini centrifuge. Carefully collect the supernatant without disturbing the bead pellet.
      NOTE: As an alternative to centrifugation, allow the beads to settle by gravity for approximately 5 min, then carefully remove the supernatant.
5. Storage
   1. Store reconstituted bo₃-LUVs at 4 °C and use them the same day for protein activity measurements.

5. Size and dispersity of LUVs by dynamic light scattering (DLS)

1. Loading LUVs for DLS measurements
   1. Transfer 45 µL of undiluted LUV or proteo-LUV suspension into a clean quartz cuvette. Ensure the cuvette is free of dust, fingerprints, or other residues to minimize light scattering artifacts.
      ​NOTE: Avoid introducing air bubbles, as they can interfere with DLS measurements. To prevent bubbles, slowly pipette the sample along the cuvette wall and do not expel the last drop forcefully. If small bubbles form, allow them to rise and dissipate before measurement.
2. Determination of LUV size and dispersity by DLS
   1. Measure the hydrodynamic diameter and polydispersity index (PDI) using a DLS instrument equipped with a 633 nm helium-neon laser and back-scattering detection at a fixed angle of 173°.
   2. Perform at least three independent measurements per sample to ensure reproducibility.
   3. Analyze the data using the general-purpose (normal distribution) model to obtain the average size and PDI.

6. Oxygen consumption analysis of LUVs

1. Temperature setup
   1. Set the measurement chamber of a Clark-type oxygen electrode system to 22 °C and allow the system to equilibrate.
2. Addition of buffer
   1. Add 1 mM Tris-HCl (pH 7.5), 200 mM sucrose buffer to the measurement chamber to a total volume of 1,000 µL. Adjust the buffer volume according to the sample type: 942.9 µL for polymer bo₃-LUVs and 953.7 µL for hybrid bo₃-LUVs.
      ​NOTE: The remaining volume will be occupied by LUVs and the DTT/Q₁ mixture to reach the final measurement volume.
3. Addition of LUVs
   1. Add bo₃-LUVs to the measurement chamber and start gentle stirring using the magnetic stirrer at 100 rpm and 22 °C. Adjust the volume of bo₃-LUVs to achieve a theoretical bo₃ oxidase concentration of approximately 27 nM: 48.6 µL for polymer bo₃-LUVs and 37.8 µL for hybrid bo₃-LUVs.
      ​NOTE: For the negative control, replace bo₃-LUVs with an equivalent volume of protein-free LUVs of the same lipid/polymer composition and concentration. All subsequent steps remain identical.
4. Baseline measurement
   1. Allow the oxygen concentration to stabilize and measure for 3 min under constant stirring at 22 °C to obtain a stable baseline.
5. Preparation of reducing/quinone mixture
   1. Freshly prepare the DTT/Q₁ mixture immediately before use by mixing 9.6 µL of 1 M DTT in Milli-Q water with 0.6 µL of 80 mM ubiquinone-1 (Q₁) in DMSO, then vortex thoroughly to ensure homogeneity.
6. Enzyme activation
   1. Activate bo₃ oxidase by adding 8.5 µL of the freshly prepared DTT/Q₁ mixture to the measurement chamber, resulting in final concentrations of 8 mM DTT and 40 µM Q₁.
7. Replicates
   1. Measure each sample three times to ensure reproducibility.
8. Data analysis
   1. Plot oxygen concentration versus time. Determine the slope of the linear portion of the curve, which corresponds to the oxygen consumption rate.
9. Reporting
   1. Report the oxygen consumption rate as the average of three measurements, with the standard deviation.

7. Preparation of protein-functionalized GUVs

1. Plasma cleaning of ITO-coated glass slides
   1. Place indium tin oxide (ITO)-coated glass slides into the plasma cleaner with the conductive (ITO-coated) side facing upward. Plasma cleans them for 1 min at high power setting ("HI") (230 V, max. RF power 18 W) and a pressure of approximately 0.5 mbar.
      ​NOTE: Successful plasma ignition can be confirmed visually by the appearance of a pink/purple glow inside the chamber.
2. Partial dehydration of proteo-LUVs
   1. Mix 20 µL of protein-free LUVs with 20 µL of proteo-LUVs containing reconstituted bo₃ oxidase-ATTO 643. Gently vortex to ensure homogeneous mixing.
   2. Deposit seven 2 µL droplets of the resulting 5 mg/mL LUV/proteo-LUV suspension onto the conductive (ITO-coated) surface of each slide used for electroformation (Figure 2A). Distribute the droplets evenly across the 160.6 mm² surface, keeping them separate to promote uniform film formation.
      NOTE: For other polymer systems, the LUV concentration and droplet volume may require optimization (typical range: 5–100 mg/mL LUVs; 0.2–2 µL droplets).
   3. Allow the droplets to partially dehydrate at room temperature (∼22 °C) and ∼20% humidity for approximately 40 min, or until a white ring forms at the droplet perimeter (Figure 2B).
      ​NOTE: Dehydration depends on ambient humidity. If the relative humidity is above 40%, extend the dehydration time as needed (up to 20 min). Determine the endpoint based on the appearance of a white ring at the droplet perimeter rather than on a fixed time. Under consistent environmental conditions (temperature and humidity), the required dehydration time is reproducible. Avoid over-drying, which is apparent as completely flat, uniformly white droplets, as this can cause protein precipitation.
3. Assembly of the electroformation chamber
   1. Assemble the electroformation chamber by sandwiching two ITO-coated slides (coated sides facing inward) separated by a 1.81 mm-thick silicone spacer with a cut on one side, which allows buffer to be added or removed during filling and collection.
   2. Fill the chamber with rehydration buffer (1 mM Tris-HCl, pH 7.5; 200 mM sucrose).
      For pH-sensitive experiments, add 10 µM pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, HPTS) to the buffer.
4. Electroformation
   1. Apply a sinusoidal alternating electric field using the following program: 50 Hz at 50, 100, 200, 300, 500, 700, and 900 mV for 6 min each; 50 Hz at 1.1 V for 2 h.
      ​NOTE: To promote the formation of larger proteo-GUVs, this step can be extended to 12 h, and 4 Hz at 2 V for 30 min for the detachment step. When the buffer contains 10 µM pyranine, increase the frequency to 500 Hz during the first two voltage ramping steps.
5. Collection of GUVs
   1. Carefully open the chamber using tweezers and collect GUVs using FineTip Ultra-Micro Pastette pipettes (or equivalent low-shear transfer pipettes) to minimize mechanical stress.
      NOTE: Avoid using standard low-volume pipette tips below 200 µL, as these can rupture larger GUVs. Alternatively, standard tips can be cut at the end to widen the opening and reduce shear during collection.
6. Storage
   1. Store proteo-GUVs at 4 °C and use within 48 h to preserve protein activity.
      NOTE: The rate of activity loss depends on the type and stability of the reconstituted membrane protein, as well as on the composition of the membrane.

8. Microscopy of protein-functionalized GUVs

1. Microscope setup
   1. Turn on the confocal microscope. If using the Leica system, launch the LAS X software. For other microscope brands, use the manufacturer’s recommended acquisition software.
   2. Select a 63× oil immersion objective (NA 1.40); adjust to a higher or lower magnification if needed, depending on the GUV size.
   3. Open two imaging channels corresponding to the fluorophores: Rhodamine Red (membrane) and ATTO 643 (reconstituted protein).
      ​NOTE: For fluorophores with partially overlapping spectra, select appropriate laser lines and narrow detection windows to minimize spectral crosstalk. Acquire each channel sequentially to prevent signal bleeding-through and ensure accurate fluorescence quantification.
2. Sedimentation of GUVs
   1. Place a press-to-seal silicone isolator onto a clean No. 1.5 glass coverslip (0.17 mm thickness).
      ​NOTE: For upright (non-inverted) microscopes, thicker glass slides may be used.
   2. Pipette 60 µL of buffer (1 mM Tris-HCl, pH 7.5; 200 mM glucose) into the center of the spacer.
   3. Gently add 20 µL of the GUV suspension on top of the buffer.
   4. Immediately cover the chamber with a second glass cover slide to avoid evaporation.
   5. Allow the GUVs to sediment for ~5 min before imaging.
3. Image acquisition
   1. Focus on the bottom of the chamber to capture cross-sections of GUVs.
   2. Acquire images of different GUVs throughout the chamber by scanning systematically from the top-left to the bottom-right corner (“snake” pattern) to obtain a representative population (~100 GUVs). Image all vesicles encountered during the scan without preselection, regardless of size or fluorescence intensity, to avoid selection bias and ensure reproducibility.
      ​NOTE: Avoid repeated imaging of the same GUV to prevent photobleaching.
4. Image analysis
   1. Quantify the fluorescence intensity of reconstituted ATTO 643–labeled protein by drawing a polyline along the membrane of individual GUVs. When using LAS X software, select Quantify → Line Profile, then choose the polyline tool and trace the membrane of each GUV. To obtain the average fluorescence intensity, open Statistics and record the mean value. Analyze only clean, unilamellar GUVs; exclude multivesicular vesicles and aggregated vesicles from the analysis.
      NOTE: If measurements are acquired on different days, fluorescence intensities should be normalized to the laser power, measured under identical imaging settings (wavelength and nominal power). To account for day-to-day variations in laser output, the following normalization is applied: , where F_a.u. is the measured fluorescence intensity in arbitrary units, P_reference is the laser power measured on the reference day, and is the laser power measured on the day of acquisition. This correction assumes a linear relationship between laser power and fluorescence intensity under non-saturating illumination conditions. In the present study, all images were acquired on the same day, and normalization was therefore not required.
   2. Report the fluorescence intensity as an average of 100 GUVs with the corresponding standard deviation.

9. Proton pumping analysis in proteo-GUVs

1. Microscope setup
   1. Turn on the confocal microscope and launch the appropriate acquisition software (e.g., LAS X for Leica systems).
   2. Select a 63× oil immersion objective (NA 1.40). Adjust to a higher or lower magnification if needed, depending on the GUV size.
   3. Configure three imaging channels corresponding to the fluorophores. For Rhodamine Red (membrane marker), use an excitation of 561 nm and an emission range of 570–620 nm. For pyranine (encapsulated pH-sensitive dye), use excitations at 448 nm and 405 nm, and collect emission across 499–551 nm.
      ​NOTE: If a 458 nm laser is available, use it instead of 448 nm to excite the deprotonated (basic) form of pyranine.
   4. Adjust laser power and detector gain to avoid pixel saturation and minimize photobleaching. Keep acquisition settings constant throughout the experiment.
2. Sedimentation of GUVs
   1. Add 280 µL of buffer (1 mM Tris-HCl, pH 7.5; 200 mM glucose) to one well of an 8-well chambered slide (total well volume: ~874 µL).
      NOTE: Do not use less than 200 µL, as volumes below 200 µL increase evaporation and pH drift. Do not exceed 400 µL to avoid spills and ensure comfortable pipetting. If using a different chamber format, adjust the volume accordingly. For inverted microscopes, use chamber slides compatible with #1.5H coverslip thickness.
   2. Gently add 20 µL of the bo₃-GUV suspension on top of the buffer without introducing air bubbles.
      ​NOTE: For the negative control, replace the bo₃-GUV suspension with an equivalent volume (20 µL) of protein-free GUVs of the same lipid/polymer composition, prepared and sedimented under identical conditions. This control accounts for any non-specific effects of DTT and Q₁ on the pyranine fluorescence ratio that are independent of bo₃ oxidase activity.
   3. Allow GUVs to sediment for approximately 5 min before imaging.
3. Baseline acquisition
   1. Focus on the bottom plane of the chamber where GUVs have sedimented.
   2. Adjust zoom to obtain 3–6 GUVs within the field of view.
   3. Record baseline fluorescence in all channels for 5 min prior to activation.
4. Activation of proton pumping
   1. Prepare a fresh DTT–Q₁ mixture immediately before use by mixing 4.8 µL of 1 M DTT with 0.3 µL of 80 mM Q₁ in DMSO, then mix thoroughly by vortexing.
   2. Activate cytochrome bo₃ oxidase by gently adding 2.6 µL of the DTT–Q₁ mixture onto the surface of the solution without inserting the pipette tip into the buffer, resulting in final concentrations of approximately 8 mM DTT and 40 µM Q₁ in the chamber.
   3. Monitor intravesicular pyranine fluorescence for 30 min under continuous acquisition.
5. Analysis of intravesicular pyranine fluorescence
   1. For each GUV, define a region of interest (ROI) within the vesicle lumen. In LAS X software, go to Quantify → Stack Profile, select Draw Ellipse, and draw a circular ROI inside each GUV.
   2. To determine the average fluorescence intensity at 499–551 nm for both excitation wavelengths (448 nm and 405 nm), open Statistics in the software and record the mean value.
   3. Calculate the fluorescence ratio as R = I₄₄₈/I₄₀₅.
   4. Plot the ratio over time to obtain kinetic traces of intravesicular pH change.

Results

The E. coli cytochrome bo₃ oxidase was expressed from plasmid pETcyo in E. coli strain C43 (DE3) ΔcyoABCDE and purified as described previously²⁹. Purified bo₃ oxidase was either used directly for reconstitution and activity measurements, or labeled with fluorescent dyes, such as ATTO 643 (this study) or other ATTO dyes^1,15. For highly hydrophilic dyes like ATTO 643, a 30 cm column ...

Discussion

Polymersomes and polymer/lipid hybrid vesicles exhibit enhanced chemical and mechanical stability compared to conventional lipid vesicles, making them highly attractive for applications ranging from drug delivery and biosensing to nano- and microreactors and artificial cells. However, their distinct membrane architecture and biophysical properties often prevent the direct transfer of protocols optimized for lipid vesicles to polymer-based systems^15,20,...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ÄKTA pure™ chromatography system	Cytiva	29018224
18:1 Liss Rhod PE	Avanti Research	810150
8 well chambered coverglass	Cellvis	C8-1.5H-N
Amicon Ultra 0.5 mL Centrifugal Filters	Sigma Aldrich	UFC5030
ATTO 643 NHS ester	ATTO-TEC	AD 643-31
Bio-Beads SM-2	Bio-Rad	1523920
Cholic acid-Na-salt (sodium cholate)	Serva	361-09-1
Coenzyme Q1	TargetMol	TGM-T19594
Confocal microscope STELLARIS 5	Leica Microsystems	603810
DTT (Dithiothreitol)	Merck	10197777001
Fine Tip Ultra Micro Pastette	Pastette	LW4243
HPTS (pyranine, 8-Hydroxypyrene-1,3,6-Trisulfonic Acid, Trisodium Salt)	Sigma Aldrich	6358-69-6
ITO-coated glass slides (25×75×1.1 mm, ≤20 Ohm/sq.)	pgo	CEC020S
LAS X software	Leica	https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/
Mini centrifuge MCF-2360	LMS	24572
Mini-extruder	Avanti Research	610000
Oxytherm+P system	Hansatech Instruments	/
Plasma cleaner (benchtop)	Harrick Plasma	PDC-32G-2
Polycarbonate membranes 0.1 μm	Avanti Research	610005
Press-to-seal silicone isolator (round, 20 mm diameter, 1 mm depth)	Grace Bio-Labs	41161502
Silicone spacers (round, 24.2 mm outer diameter, 14.3 mm inner diameter, 1.8 mm depth)			laboratory-made, cut from silicone sheet
Soy PC (95%)	Avanti Research	441601
Specord 50 PLUS spectrophotometer	Analytik Jena	823-0200P-3
Superdex 200 10/300 GL column (column L × I.D. 30 cm × 10 mm)	Cytiva	28-9909-44
Test tube shaker (2800 rpm)	IKA Lab Dancer	3365000
Thermomixer comfort 5355	Eppendorf	5355000.011
Xiameter OFX-5329 (PDMS-g-PEO; MW 3000)	Dow Corning	000000000004109892
Zetasizer Nano ZS	Malvern Panalytical	ZEN3600