The overall goal of this gel-assisted rehydration method is to robustly form intact unilamellar giant polymer vesicles in which vesicle size distribution can be easily controlled. This method offers a robust alternative for the formation of polymer vesicles, which can be used to investigate the physical properties of the membrane and then integrate those vesicles into bioengineering applications. The main advantage of this technique is the ability to easily produce hundreds of intact giant polymer vesicles much more quickly and in a less labor intensive manner than traditional techniques.
So, the idea for this came about when we learned of the gel-assisted rehydration technique for forming giant lipid vesicles. Our current methods for forming giant polymersomes, however, are challenging and often low-yielding, so we decided to use this methodology as an alternative approach. Start the polymer solution preparation by swirling five milligrams of PEO-PBD diblock copolymer in one milliliter of chloroform in a glass vial until fully dissolved.
Add a five milligram per milliliter solution of polymer and chloroform and a one milligram per milliliter solution of fluorescently labeled lipid and chloroform to a new vial until a final concentration of 99.5 mole percent polymer and 0.5 percent lipid is obtained. To minimize evaporation, transfer the polymer solution to an airtight vial with a chloroform resistant lid, and store it at 20 degrees Celsius for later use. To begin agarose film preparation, add 50 milliliters of water and 0.5 grams of standard agarose to a 250 milliliter Erlenmeyer flask.
Boil for one minute using a microwave. Before continuing, cool the agarose solution to between 65 and 75 degrees Celsius to prevent poor adherence or clumping. Next, cut the end off of a 1000 microliter pipette tip and use it to pipette 300 microliters of the agarose solution onto a 25 milliliter square glass cover slip.
While wearing gloves, hold the edge of the cover slip and spread the agarose evenly across the entire surface using the long edge of another 1000 microliter pipette tip. Place the cover slip agarose side up onto parafilm and incubate it at 37 degrees Celsius for at least one hour. Continue by pipetting 30 microliters of the previously prepared polymer solution onto the agarose film coated cover slip.
Hold the cover slip edge with gloved fingers and spread the polymer solution over the agarose film in a circular motion using the long edge of an 18 gauge needle. Finish by placing the cover slip polymer side up in a plastic petri dish inside a vacuum dessicator. Evacuate the dessicator for at least one hour to remove any residual solvent.
To form polymersomes, first adhere a cover well to the polymer side of the coated cover slip by pressing gently until a tight seal is formed. Then, add 200 to 500 microliters of rehydration solution to the chamber. Next, place a wet rolled lab wipe along the inside edge of a glass petri dish to form a humidity chamber.
Place the cover slip with the adhered cover well into the dish and cover it with a lid. Cover the petri dish with foil to minimize photo bleaching and then set it on a hot plate at 40 degrees Celsius for at least 30 minutes to complete polymersome formation. To begin the FRAP imaging process, place the cover slip with the adhered cover well onto the stage of an inverted fluorescence microscope fitted with an adjustable condenser.
Next, select a filter set based on the fluorescent lipid used and to image the polymersomes on the top surface of the cover slip using a 100X oil objective. To characterize membrane fluidity using FRAP, first focus on a polymersome, then close the microscope condenser to a small aperture and ensure that the edges of the polymersome lie within the image field. Subsequently, increase the camera exposure, remove all the neutral density filters and allow the region of interest to photo bleach for 30 to 60 seconds.
Finally, turn off the lamp and fully open the condenser. Decrease the exposure to the starting settings and capture images every 30 seconds for three minutes. Open the image analysis software on the microscope's computer and load the acquired images of the polymersomes.
Set the scale for the measurement by opening the Analyze drop down menu and selecting the Set Scale option. Enter the appropriate calibration units for distance in pixels, known distance and unit of length, and then select okay. Next, use the line toolbox and draw a line spanning the diameter of a selected polymersome.
Measure the diameter of the polymersome by opening the Analyze drop down menu and selecting Measure. To aid analysis, use control plus D to print the measurement line on the image before moving to the next polymersome. Continue measuring polymersomes using the above procedure and adding each measurement to the data window until complete.
Finally, save the data by clicking File and Save As.Click Save in the window that opens and then open the results file as a spreadsheet to analyze the data. Polymersomes formed from PEO-PBD polymer films rehydrated with various buffers were imaged using fluorescence microscopy. The images show the polymersomes to be non-homogeneous in size under the different rehydration conditions with the diameter typically under 10 micrometers.
Epifluorescence images of polymersomes formed in water at different temperatures show that as rehydration temperature increases, larger polymersomes are formed. The average diameters for greater than 100 polymersomes formed in water at different rehydration temperatures were calculated. At 24 degrees Celsius, the average diameter was 2.93 micrometers, increasing all the way up to 14.04 micrometers at 70 degrees Celsius.
Once mastered, this technique can be done in approximately one hour if performed correctly. While attempting this procedure, it is important to keep the polymersomes in solution at all times. Make sure that the cover well is securely attached to the cover slip to avoid leakage of the rehydration solution and drying of the polymersomes.
Following this procedure, methods such as electron microscopy can be used to understand additional structural properties of the polymersomes. This technique paves the way for research in synthetic biology and bioengineering to explore polymersomes as a robust, chemically tailorable alternative to lipid membranes for research and abiotic-biotic interface, drug delivery research and protocell systems. After watching this video, you should have a good understanding of how to robustly form giant polymersomes using gel-assisted rehydration, tune the overall size of the vesicles and characterize their membrane integrity.
Don't forget that working with chloroform or the agarose and needles can be hazardous. Pay close attention while performing these procedures. Take precautions such as working in a fume hood and use goggles and gloves at all times.
