A Thermoplasmonic Approach for Investigating Plasma Membrane Repair in Living Cells and Model Membranes

1. Cell membrane puncture preparation

1. Cell seeding (Day 1)
   1. Culture human embryonic kidney (HEK293T) cells in culture medium at 37 °C in a 5% CO₂ humidifier incubator until they reach 70% confluency.
   2. Detach the cells from the surface using 500 µL of trypsin, count 200,000 HEK293T cells, and seed them in a culture dish with a total volume of 3 mL of culture medium. Incubate the cells at 37 °C in a 5% CO₂ humidifier incubator for 24 h.
      NOTE: To prevent cell clustering in the center of the dish, spread the cells evenly and avoid swirling the dish, as this can decrease transfection efficiency.
2. Cell transfection (Day 2)
   NOTE: The transfected cells can be used up to 48 h after transfection.
   1. Pipet the plasmid of interest and the transfection reagent for 5 s before use.
   2. In a sterile 2 mL tube, mix the following in the specific order: 500 μL of reduced-serum medium, 5 μL of transfection reagent (4 times more than the plasmid), and 1.25 μL of plasmid (1 μg/μL).
      NOTE: To investigate calcium influx upon membrane rupture, follow the same procedure but use the membrane-bound calcium sensor probe GCaMP6-CAAX (1 μg/μL).
   3. Gently but thoroughly pipet the transfection mixture and incubate it at room temperature (RT) for 30 min before adding it dropwise to the cells.
   4. Before adding the transfection mixture, remove the medium from the culture dish, gently wash the cells with 2 mL of phosphate-buffered saline, and add 2000 μL of reduced-serum medium to the dish.
   5. Incubate the cells together with the transfection mixture at 37 °C in a 5% CO₂ humidifier incubator for 2 h and 45 min before changing the medium to 3 mL of culture medium.
3. Preparation of gold nanoparticle (AuNP) solution (Day 2)
   1. Vortex the 200 nm AuNP stock solution for 10 s at level 10 (see Table of Materials for further specification of apparatus), sonicate for 5 min (max amplitude), and vortex again for 10 s.
   2. Mix 150 μL of AuNPs with 850 μL of distilled water to a total volume of 1 mL.
      NOTE: The diluted AuNP solution can be stored in the fridge and reused for up to 1 month.
4. Preparation of the experimental dish (Day 2)
   1. Coat the microwell dish with 150 μL of 0.01%-0.1% cell attachment solution and incubate for 15 min at RT.
   2. Wash the glass surface twice with 500 μL of distilled water and allow it to air dry for ~10 min.
   3. Add 80 μL of AuNP solution dropwise to the dry surface.
      NOTE: Vortex (10 s), sonicate (5 min), and vortex (10 s) the diluted AuNP solution before adding it to the coated glass surface to minimize AuNP aggregates.
   4. Wait for ~10 min before introducing 1.5 mL of culture medium. Let the dish incubate at 37 °C overnight.
5. Preparation of the experimental chamber (Day 3)
   ​NOTE: The experimental chamber can be prepared on either day 3 or 4; however, ensure that the following preparations are done on the same day as the experiment.
   1. Remove the medium from the cells in the culture dish and wash the cells with 2 mL of phosphate-buffered saline.
      NOTE: This step is essential to remove any residual medium and debris that could interfere with the subsequent steps.
   2. Add 500 μL of enzyme-based cell detachment solution to the well and incubate for 1-3 min until the cells have detached from the culture dish.
   3. Add 1.5 mL of fresh culture medium and pipette the cell solution to get a homogenous cell solution to minimize the likelihood of cell clusters.
   4. Carefully remove the medium from the AuNPs in the experimental microwell.
   5. Add the cell solution (2 mL) to the microwell and allow it to incubate for at least 5 h before performing the experiment.
      NOTE: For optimal experimental conditions, avoid swirling the chamber, as this may cause cells to cluster in the middle of the chamber.

2. Cell membrane puncture experiment

1. Optical settings of the experiment
   1. Conduct the experiments using a confocal scanning microscope combined with a 1064 nm trapping laser⁵⁷.
   2. Carry out the optical trapping at the focal plane utilizing a 63x water-immersion objective with a numerical aperture (NA) of 1.2.
   3. Assume that the focus is the size of an Airy disc and that the focal beam width of the irradiation laser is ~540 nm in radius.
   4. Convert the laser power (P) to the corresponding laser intensity (I) by calculating the laser power per area (W/cm²).
   5. Utilize an acousto optical beam splitter (AOBS) to visualize multiple fluorescent signals detected using photomultiplier tubes and simultaneous detection of metallic NPs via their scattering signal.
      NOTE: Not all confocal systems are equipped with AOBS, which allows reflection imaging of metallic NPs. Here, other forms of detection must be implemented, or a sequential scanning of the NIR laser in the area underneath the cell can be attempted.
   6. During the experimental sessions, mount a glass-bottom open chamber containing cells, molecular fluorescent probes, and gold nanoparticles on the microscope.
   7. Move the trap relative to the cells by translating the sample mounted on a piezoelectric stage, enabling nanometer-precise lateral movements¹⁶.
      NOTE: The optical trap is kept stationary.
2. Experimental settings for the cell membrane puncture
   1. Place HEK293T cells, transfected with annexin plasmids coupled with a fluorescent protein, on top of isolated AuNPs that are immobilized on the glass surface (Figure 1A).
   2. Utilize an Argon 488 nm laser to visualize the GFP fluorescence signal and a HeNe 633 nm laser for observing AuNP reflection in the scanning confocal microscope.
   3. Employ the optical tweezer, which operates with a 1064 nm NIR laser, to irradiate a single AuNP for ~1 s, inducing a significant local increase in temperature that disrupts the plasma membrane.
   4. Apply irradiation between 200-295 mW to the focused particle, resulting in a substancial temperature increase.
      NOTE: There is a substantial loss of power within the optics, with the laser power at the focal point reaching around 20% of the stated milliwatt, depending on the specific setup. The specific intensity (measured in W/cm²) depends on the exact alignment of the system, specifically the focal size. Additionally, the heat conduction of glass is higher than that of cell and water and consequently reducing the amount of heat released to the plasma membrane is slightly reduced^48,58.
   5. Achieve effective and localized PM injury and subsequent protein repair response by properly calibrating the localization of the NIR laser focus spot. This is achieved by capturing a single AuNP suspended in the same imaging medium and ensuring that the selected AuNP is in focus prior to irradiation.
      NOTE: The nanoparticle is considered to be in focus when its scattering signal appears the sharpest, i.e., exhibiting clear edges and the absence of a halo around the particle, when observing it in confocal microscopy (Figure 2C (ii)).
3. Cell and AuNP density conditions
   1. Choose single cells instead of cell clusters to prevent overlap of plasma membranes.
      NOTE: The cells should be adhered to the surface, exhibiting a flattened morphology (Supplementary Figure 1), which allows puncturing of the cell periphery while preventing damage to the nuclear membrane (Supplementary Figure 2).
   2. Incubate the cells according to the protocol or until they have settled and flattened to prevent AuNP cellular uptake. Avoid excessive incubation time and reduce the likelihood of AuNP endocytosis using PEGylated AuNPs.
   3. Ensure that the immobilized AuNPs are present as single particles, adequately spaced apart from each other to prevent aggregates. Aggregates can lead to a significant increase in the thermal gradient, resulting in elevated temperatures that may disrupt a substantial fraction of the cell.
   4. Replace the sample every 1-2 h to maintain cell health.
      NOTE: Prolonged exposure can lead to a deterioration in cell health, compromising their ability to respond accurately to injury in terms of membrane repair. This decline in cell health is typically observed by a change in cell shape, as cells appear more spherical and more rigid, eventually culminating in cell death.

Supplementary Information. Please click here to download this File.

3. Giant unilamellar vesicle (GUV) preparation

1. Preparation of the lipid mixture
   1. Make the GUV lipid composition by combining 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) lipids in a molar ratio of 4:1 (see Table 1). Aliquot the lipid stock into 1.5 mL glass vials according to the experimental needs and store them at -20 °C.
      NOTE: For prolonged lipid preservation and to prevent the oxidation of unsaturated lipids, replace the air with argon in the aliquoted vials.
   2. Before mixing the lipids, thoroughly clean a 50 μL and a 500 μL glass or metal syringe with chloroform five times to ensure they are free of contaminants for the chloroform-dissolved lipids.
      CAUTION: Handle chloroform in a fume hood due to its toxicity.
      1. Transfer the calculated volume of chloroform into a clean amber-glass vial, followed by the specified amount of each lipid (see Table 1).
         NOTE: To avoid cross-contamination between lipid stocks, ensure that the syringes are cleaned with chloroform.
      2. Finally, add the membrane dye and thoroughly mix the lipids by pipetting. Store the prepared lipid mixture at -20 °C for further use; the mixture remains viable for 2-3 weeks without any considerable lipid damage.
         NOTE: The mixing should be done with a metallic or glass syringe. Always keep the lipids on ice when they are out of the freezer.
2. Preparation of polyvinyl alcohol (PVA) gel
   1. Prepare the GUVs using the gel-assisted hydration method described by Weinberger et al.²³ with minor modifications.
      1. Prepare the PVA gel by dissolving 5 g of PVA in 100 mL of a buffer containing 50 mM sucrose, 25 mM NaCl, and 25 mM Tris (pH 7.4).
      2. Heat the PVA solution to 85 °C and stir until it becomes transparent. Allow it to cool down to RT and store it in a fridge for further use.
         NOTE: PVA is not properly dissolved in the buffer, and therefore, heating up to 85 °C is necessary.
3. Preparation of glass slides
   1. Clean glass slides with ethanol and let them air-dry. Then, treat the slides with an air plasma cleaner to remove any residual contamination from the glass surface.
   2. Preparation of PVA-coated glass slides
      1. Warm the PVA gel (5%) up to 60 °C for 30 min to increase its fluidity. Apply 90 μL of the warm PVA onto the glass slide, spread uniformly, and let it dry in a heating cabinet at 50 °C for 50 min.
      2. Once the PVA glass slide is ready, add 30 μL of the prepared lipid mixture using a glass or metal syringe and spread it into a thin film using the edge of the needle.
      3. Dry the lipid mixture by evaporating its chloroform content under gentle nitrogen pressure. Further, dry the glass slides under a vacuum for 1.5-2 h.
4. Growing GUVs in a chamber
   1. Assemble the in-house chamber, similar in design to a previously published report⁵⁹, using the prepared glass slide.
   2. In a separate 2 mL tube, dilute the recombinant protein of interest (in this case, ANXA5 or ANXA4) to a final concentration of 500 nM with a growing buffer (GB) consisting of 80 mM sucrose, 70 mM NaCl, and 25 mM Tris-HCl (pH 7.4).
   3. Add 400 μL of the diluted recombinant protein solution to the chamber. Incubate the chamber at RT for 1 h to allow GUVs to grow from the deposited lipid coat. Use the same buffer, excluding the protein, as a negative control.
      ​NOTE: Wrap the chamber in polyolefin film to prevent buffer evaporation.
   4. Collect the GUVs by transferring 400 μL of the chamber content to a 2 mL tube.
   5. Remove non-encapsulated proteins outside the GUVs by adding 1 mL of observation buffer (OB) containing 55 mM glucose, 70 mM NaCl, and 50 mM Tris-HCl (pH 7.4) to the collected solution. Then, centrifuge the solution at 600 x g for 10 min at 13 °C.
   6. After centrifugation, replace 1 mL of the supernatant with an equal volume of observation buffer. Disperse the GUVs through gentle pipetting and store them in the refrigerator until they are used in GUV experiments.

4. GUV puncture experiment

1. Chamber preparation
   1. Use a 35-mm glass-bottom dish for the experiment. To prevent GUVs from sticking to the surface and bursting, coat the surface with β-casein (5 mg/mL) for 15-30 min.
      1. For the β-casein solution, dissolve 0.1 g of the protein in a 20-mL buffer of 20 mM Tris (PH 7.5) and 100 mM NaCl. Filter the protein solution, aliquot it into small vials, and freeze it for further use.
   2. Wash the chamber twice with observation buffer to remove any excess free β-caseins from the surface and let dry at RT.
   3. In a separate 2 mL tube, mix the collected GUVs with OB. Add CaCl₂ to the mixture for the desired final concentration (in this case, 200 μM).
   4. Introduce 150 nm gold nanoshells (AuNSs) to the mixture in a ratio of 1:100. The final mixture comprises 250 μL of GUVs, 225 μL of OB, 20 μL of CaCl₂ (5 mM), and 5 μL of the specified AuNSs.
2. Experimental setup
   1. Transfer the mixture to the chamber and mount it on the microscope stage. Depending on the size of the chamber, add either the entire mixture or a portion of the mixture.
      NOTE: Timing is critical since calcium ions can pass through the membrane and mediate the binding of the annexins to the internal leaflet of the membrane.
   2. Employ the same optical setup used for the cell puncturing experiments.
   3. Use the optical tweezers to trap an individual AuNS in close proximity to or on the surface of a GUV by applying a laser power of ~ 125 mW.
   4. Subsequently, increase the laser power to ~ 300 mW. This produces a highly localized temperature increase, which disrupts and punctures the membrane at the target site.
      ​NOTE: AuNSs are preferred for the GUV experiments due to their ability to generate a higher transient temperature increase while maintaining a smaller size compared to solid AuNPs.

Table 1: Table for determining the GUV composition. Please click here to download this Table.

5. Measurements and data analysis of ANXA response in cell puncture experiments

1. Use MATLAB to analyze the images and to calculate and plot the AuNP temperature profile (Figure 1D, E) based on Mie's equations^60,61.
2. Additionally, process all confocal images using the FIJI ImageJ distribution^62,63.
3. Calculation of the ANXA ring radius
   1. Crop the region of interest containing the punctured area from the raw data prior to membrane injury analysis.
   2. Employ the in-house MATLAB workflow for processing each image by manually marking the center of the ANXA ring and its outer perimeter.
      1. Use these markings to set the processing limits for the region of interest.
         NOTE: The area is subsequently processed radially within the set limits, and the fluorescence intensity at a specific distance to the center is the average over the full circle with the same center distance. This number is stored for each radius step.
      2. Use the workflow to fit the averaged intensities over the entire scanning range to a one-dimensional Gaussian curve to identify the maxima (R_p) and full width at half maximum (FWHM) corresponding to R_ext and R_int, respectively, as depicted in Figure 3A.
   3. Finally, determine the radius of the ANXA ring by the distance from the ring's center to R_ext given by the plot generated by the MATLAB workflow.
4. Three-dimensional ANXA ring radius calculation
   1. Track the change in ANXA ring radius in the z-direction using the same MATLAB analysis workflow as in step 5.1. Apply the workflow to several confocal z-sections across the membrane wound, as illustrated in Figure 3C, E.
   2. Calculate the slope for each wound based on the change in radii over the z-sections, as depicted in Figure 3F.
5. Time evolution of the ANXA ring radius
   1. Similarly, track the ANXA ring's evolution of the ANXA ring after thermoplasmonic membrane puncture using the MATLAB analysis workflow from step 5.1. Analyze consecutive time points to monitor changes over time, as demonstrated in Figure 3D, G, H.