Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

The overall goal of the following experiment is to present a robust preparation method for biologically relevant lipid vesicles. This is achieved by mixing and transferring phospholipids to glass vials and hydrating them with aqueous buffer to create a multi lamella SLE suspension. As a second step, a freeze and thaw method is performed, which subsequently results in the production of Ella Vesicles from multi lamella vesicles.

Next, the liposome suspension is passed through an extruder using the determined optimal pressure to produce homogeneous lipid vesicles of a desired size. Results were obtained that show consistent submicron vesicle sizes based on dynamic light scattering and nanoparticle tracking analysis. The main advantage of this technique over existing methods like sonication and sedimentation, is that this method uses a pressure control system with optimal determined nitrogen flow rates for efficient lipid vesicle size preparation.

To begin this procedure, retrieve a 20 milliliter glass vial with a Teflon lined cap, clean all glassware and syringes with chloroform prior to use to prevent contamination. Transfer 100 microliters of reagent grade chloroform to the glass vial using a 250 microliter airtight glass syringe. Add 30 microliters of reagent grade methanol to the same glass vial using a 100 microliter airtight glass syringe.

Prepare a two millimolar pop C poppy cholesterol lipid solution by transferring 216 microliters of 10 milligrams per milliliter pop C 42 microliters of 10 milligrams per milliliter. Pop e and 20 microliters of 11 milligrams per milliliter. Cholesterol solutions to the 20 milliliter glass vial evaporate the organic solvents using slow flow argon or nitrogen gas until a thin film of lipids is observed on the bottom of the vial, place the uncapped glass vial in a vacuum desiccate for at least 30 minutes.

To remove residual solvent, transfer two milliliters of buffer previously passed through a 0.2 micron filter to the glass vial to hydrate the lipids. Incubate the mixture at four degrees Celsius overnight and use within 48 hours for liposome sizes of 30 to 100 nanometers. Free the liposome suspension in liquid nitrogen for 15 seconds.

Then thaw the liposome suspension using a heating block at 42 degrees SIUs for approximately three minutes. Repeat the freeze thaw process for a total of five cycles. Freezing and subsequently thawing produces Ella Les from multi lamella vesicles and enhances lipid trapping efficiency during extrusion.

Next, prepare for extrusion by following the Avastin instructions to carefully assemble the lipo so fast. LF 50 extruder correct assembly is critical to prevent nitrogen leakage, which affects desirable flow rates. To ensure success, perform the following steps carefully.

To produce an airtight seal. Place the large hole support screen in the support filter base followed by the circular center dish, one drain disc, and one polycarbonate membrane. Place the small black O-ring on top of the membrane and secure it to the support filter base.

Attach the top filter extruder by tightening four screws in the four corresponding holes. Assemble the filter extruder unit underneath the large extruder barrel. Add the liposome solution to the cylinder barrel on top of the extrusion barrel.

Place a large circular O-ring, narrow cap, large circular O-ring and circular cap. Attach the gas regulator to the extruder barrel top and close all valves to prevent air leakage, including the pressure relief valve. Place a 20 milliliter glass vial or a 50 milliliter erlenmeyer flask under the extruder filter unit.

A safety valve is connected to the regulator and will release if pressure exceeds 600 PS.I turn on the nitrogen gas and open the gas valve connected to the extruder. Increase nitrogen pressure to 25 PSI for 400 nanometer liposomes to 125 PSI for 100 nanometer liposomes, and to 400 to 500 PSI for 30 nanometer liposomes. Watch the liposome suspension until it ejects into the container while being pushed by the nitrogen.

Keep the nitrogen flowing until no liquid is observed. Passing through the extruder filter into the glass file to perform DLS analysis. First, prepare 50 microliters of a 20 micromolar liposome solution diluted in phosphate buffered saline or PBS.

Then turn on the power source and the lamp source. Proceed to open the dynapro software. Set the software to the algorithm model to detect liposomes at the desired size.

After connecting to the hardware pipette 14 microliters of liposome sample into the quartz vete and insert into the cell holder, press start following approximately 20 to 30 acquisitions, press stop. Analyze the average liposome diameter peaks recorded on the histogram. Nanoparticle tracking analysis begins with the preparation of a 500 microliter 0.1 micromolar liposome solution solubilized in PBS, rinse the sample compartment with water and ethanol.

Then dry the sample compartment with a lint-free paper towel. Turn on the laser power source and the computer deliver 300 microliters of 0.1 molar liposome solution to the sample compartment. Open the temperature control and the nanoparticle tracking analysis software.

Press the capture button to turn on the laser. Use the horizontal and vertical adjustments to move the stage and adjust the microscope focus. Set the desired temperature and recording time.

Press the record button to take multiple picture frames of the liposome particles for a specified amount of time. Analyze the peaks corresponding to the diameter liposome sizes on the histogram as it tracks the motion of each particle. DLS is an established method that collects scattered light to determine the particle diameter and was performed to determine the liposome sizes.

Hydrated liposomes were extruded through polycarbonate membranes at various sizes and pressures as described in the written protocol. The diameters of the liposomes measured by DLS for 30 nanometer pore size was 66 plus or minus 28 nanometers. For 100 nanometer pore size was 138 plus or minus 80 nanometers, and for 400 nanometer pore size was 360 plus or minus 25 nanometers.

A suspension of 50 nanometer polished Irene beads was used as a calibration standard where it recorded a diameter of 47 plus or minus 16 nanometers. The percent poly dispersity shows that there is no overlap within liposome sizes. It is typical to observe diameters higher than 30 nanometers when using DLS analysis due to the known bias this instrument has towards larger particles.

Despite this instrumental limitation, the calibration curve describes alinear correlation. Nanoparticle tracking analysis is new technology that measures the size of each particle from direct observations of diffusion in a liquid medium independent of particle refractive index or density. This high resolution technique can be used to supplement the measurement of liposomes with DLS, the NTA recorded diameters of 95 plus or minus 48 nanometers and 356 plus or minus 51 nanometers for two 100 nanometer and 400 nanometer polystyrene solutions were used for calibration.

Lipid solutions have a more comparable diameter relative to DLS producing average sizes of 29 plus or minus 14 nanometers for 30 nanometer pore size, 95 plus or minus 17 nanometers for 100 nanometer pore size and 359 plus or minus 73 nanometers for 400 nanometer pore size. NTA may be a more general characterization technique to quantify microscopic particles since its sensitivity allows for the measurements of particles of 50 to 1000 nanometers. The calibration curve shows a linear correlation between the polycarbonate membrane pause versus the recorded NTA diameter negative stain transmission Electron microscopy images show each liposome size following extrusion sizes 3, 100 and 400 nanometers a clearly distinct from each other.

The magnification was set to 25, 000 times with the scale bar representing 0.5 microns. A time course experiment was conducted with three liposome sizes. DLS was measured for each liposome solution immediately following extrusion.

All liposome solutions were stored at four degrees Celsius overnight. Their diameters were recorded again by DLS after an overnight incubation. Little to no change was observed after a 60 hour incubation period While attempting this procedure, it is important to remember that multiple passes through the extrusion filter will improve Vesco solution homogeneity.

Also, remember to use glass vials and syringes for sample preparation to prevent contamination through leaching by polypropylene tubes from chloroform.