The construction of lipid bilayer membranes described in this protocol can be used to obtain crucial information about membrane interactions with pharmaceutical agents, environmental toxicants, and even biological compounds. These techniques can be used to fabricate membranes of varying liquid complexity, and to assess compound permeability, absorption, and embedment within membranes. To begin, add the appropriate volume of lipid stock solution into a clean glass vile to achieve a final vesicle concentration of 2.5 milligrams per milliliter after rehydration.
Remove chloroform from lipid solution, using a stream of nitrogen gas. To ensure full removal of chloroform, connect the dried lipid film to the vacuum for at least four hours. Rehydrate the dried lipid film with a required volume of Tris sodium chloride buffer to yield the final vesicle concentration of 2.5 milligrams per milliliter, and vortex for approximately 15 to 30 seconds.
Transfer the vesicle suspension into a container with dry ice until frozen for approximately 30 minutes. After the sample is completely frozen, thaw the suspension in a 30 to 40 degree Celsius water bath. Vortex the thawed vesicle suspension.
To make one milliliter or less of vesicles, obtain a mini extruder. Pre-wet a filter support with ultra pure water, then place it on the membrane support surface inside the O ring. Repeat for the second internal membrane support.
Position one internal membrane support into the extruder outer casing. Place one 100 nanometer polycarbonate membrane onto the internal membrane support directly over the filter support. Position the second internal membrane support into the extruder outer casing with the O ring and filter support side facing the polycarbonate membrane.
Attach the polytetrafluoroethylene bearing into the retainer knot and screw it closed with the extruder outer casing. Clip the extruder into the heating block. Load the lipid vesicle suspension into one of the syringes and position the syringe into the extruder heat block, inserting the needle fully into one end of the extruder.
Insert the second empty syringe into the opposite side and lock both syringes using the arm clips on the heat block. Slowly push the vesicle suspension into the empty syringe and then back into the original syringe. Repeat 20 more times for a total of 21 passes through the polycarbonate membrane.
Transfer the lipid vesicles into a clean glass vile for storage. To extrude five to 50 milliliters of vesicles, assemble the large extruder by placing the large hole screen support, centered disc, drain discs, and polycarbonate membrane into the space in the extruder lower support. Connect the extruder upper and lower supports using the four screws and tighten.
Attach the extruder unit to the sample cylinder by scrubbing it to the bottom and tightening with a wrench to secure. Fill the sample cylinder with ultrapure water and extrude the water through the extruder unit prior to adding the sample into the sample cylinder. Add the lipid vesicle suspension into the sample cylinder and screw the top closed.
Slowly increase the pressure until the sample begins to drip from the extruder unit at a rate of approximately two to three drops per second into a clean glass vile. Once all sample has been extruded, turn off the nitrogen supply and release the pressure in the sample cylinder by opening the pressure relief valve slowly. Pour the lipid vesicles back into the sample cylinder and repeat previous step nine more times for a total of 10 extrusions.
Load the vesicle suspension into in appropriate cuvette, and insert into the dynamic light scattering instrument, input the sample details, and perform the measurement using the associated software. Place the zeta cell in the sample chamber, ensuring that the electrodes are in contact, and close the sample holder lid. In the associated software, input the sample details and collect measurements.
Insert the silica coded quartz crystal sensor into the flow module, ensuring that the T shape on the crystal aligns with the T shape on the module and screw the flow module closed. In instruments with multiple chambers, additional bilayers can be formed in parallel. Connect the inlet and outlet tubing to the flow module and pump.
Place the tubing into the holding guards and close the lid of the analyzer system. Place a waste container at the outlet of the pump to collect spent solutions. To perform the cleaning, first turn on the pump and set the flow speed to 400 microliters per minute.
Insert the inlet tubing into ultra pure water and flow five to 10 milliliters through the module. Switch tubing into SDS and flow five to 10 milliliters through the module. Switch tubing back into ultra pure water and flow 10 to 20 milliliters through the module.
Finally, flow air through the module until no remaining solution is collected. Remove the sensor from the flow module and rinse the sensor with ultra pure water. Drive the sensor and the flow module with a nitrogen gas stream, making sure that the electrode always remains free of any liquid.
In a chemical fume hood, insert the silica coated quartz crystal sensor into an ultraviolet or ozone cleaning instrument. Turn on the instrument and allow treatment for at least two minutes. Remove the sensors carefully and return them into the flow module.
Turn the analyzer instrument on to connect the associated software and set temperature to the desired value for forming the supportive lipid bilayer. Allow temperature to stabilize to the desired input. Configure the measurement and find all sensor resonance frequencies, and dissipation for overtones three, five, seven, nine, 11, and 13 before starting the measurement.
Allow Tris sodium chloride to flow through the module for five to 10 minutes, ensuring that the baseline frequency change or Delta F and dissipation change or Delta D values in liquid remain stable. Stop the pump, remove the inlet tubing from the Tris sodium chloride solution, and carefully insert it into the lipid vesicle solution. Backflow for five seconds to remove any air bubbles from the inlet tubing, and then continue the forward flow.
Restart the measurement in the software to zero the baseline. Flow lipid vesicles until bilayer formation is observed in real time in the data acquisition software. Then repeat this step to change the inlet tubing from lipid vesicles back to Tris sodium chloride buffer.
Add five microliters of the lipid solution to the donor compartment, which is a porous polyvinylidene difluoride 96 well multi-screen filter plate with 0.45 micro meter pore size. Immediately submerge the filter plate into the acceptor compartment, which is a transport receiver plate containing 300 microliters of phosphate buffered saline. Add 200 microliters of phosphate buffered saline to the transport donor compartment.
Follow previous steps of stopping the pump and changing the inlet tubing to lipid vesicle solution and observing bilayer formation, then change the inlet tubing into the alpha helical or AH peptide solution. Introduce the solution into the flow module until frequency change and dissipation change are observed from the new solution addition. Stop the pump and allow the AH peptide to incubate with the vesicles for 10 minutes.
Switch the inlet tubing into Tris sodium chloride, and start the flow to remove the AH peptide from the ruptured vesicles, leading to successful formation of a lipid bilayer. First, flow the molecule solvent, then, switch the inlet tubing into the solution containing the molecule of interest and flow for at least five minutes. If desired, stop the flow and allow the liquid containing the desired molecule to incubate with the bilayer.
Change the inlet tubing back to the molecule solvent alone. Flow for at least five minutes, then switch the inlet tubing into Tris sodium chloride, and flow for at least five minutes. In the software, stop the measurement, save the file, and stop the pump.
Immediately following previous steps for a uni-lipid suspended bilayer or for a multi-lipid suspended bilayer, remove PBS from the donor filter plate compartment and replace with 200 microliters of the test solution. Immediately submerge in a new transport receiver plate with 300 microliters of PBS. After incubation with gentle rocking for two hours at 25 degrees Celsius, collect 150 microliters of the solution from the donor and acceptor compartments.
Measure the molecule concentration in both samples using an appropriate method based on properties of this molecule. Size, polydispersity, and Zeta potential of Uni-lipid egg PC vesicles, and two multi lipid vesicle compositions were compared. The size distribution of the egg PC vesicles and multi lipid one vesicles were nearly identical.
Both egg and PC multi-lipid one vesicles were negatively charged, while multi-lipid two vesicles were positively charged. Uni-lipid egg PC vesicles readily absorbed to the surface, as shown by the decrease in frequency change, and increase in dissipation change, followed by spontaneous rupture. Multi-lipid vesicles absorbed to the surface, but require the addition of AH peptide to rupture the vesicles, resulting in lipid bilayer formation.
With supported lipid bilayers, frequency change and dissipation change can be analyzed before and after introduction of a compound of interest. For example, interactions of DEHP and environmental toxicant with supported uni and multi-layered by layers were investigated. Similar levels of DEHP absorption were observed for both lipid bilayer types.
However, differences in dissipation change were observed between the bilayers with a larger dissipation change seen for the egg PC bilayer compared to the multi lipid one bilayer. Little permeation was observed for both uni-and multi-lipid one bilayers. Following this procedure, a variety of cell mimicking lipid bilayers can be developed to enable cell-free screening of molecular interactions with compounds ranging from pharmaceuticals to environmental toxicants.
