Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

Microfluidics is an emerging technique for preparing liposomes of a tunable nanoparticle size and with great reproducibility and scalability. This protocol enables the continuous high through output production of low temperature sensitive liposomes for co-loading with a chemotherapeutic drug such as doxorubicin and a fluorescent dye such as indocyanine green. Cell preparation primarily use a top down approach such as lipid film hydration and extrusion.

It remains challenging to prepare large and reproducible batches for clinical applications. A major advantage of microfluidics is the ability to handle small liquid volume with high controllability in space and time while operating in a continuous and automated manner. Demonstrating the procedure with Calvin Cheung will be Guanglong Ma and Amalia Ruiz, the post-doctoral researchers from my laboratory.

To assemble the syringe pumps, use the pump-to-pump network cable to connect the to computer port of the secondary syringe pump to the to network port of the master syringe pump. Use the PC to pump network cable to connect the to computer port of the master pump to the RS-232 serial port of the computer. To assemble the staggard herringbone micromixer microfluidic device, use a nut and ferrule to connect tubing to each of the inlets and outlets of the device.

Then use a second nut and ferrule and a union assembly to convert the terminal of the tubing for both inlets to a female luer. To set up the pump control software, use the setup button of the syringe pump to assign the address of the master syringe pump and secondary syringe pump to Ad:01 and Ad:02 respectively, then open the pump control software on the computer. The two syringe pumps should be detected automatically followed by a beeping sound.

Select HSW Norm-Ject 5cc diameter equals 12.45 to assign the diameter to 12.45 millimeters. Set the rate to 0.25 milliliters per minute for pump one and 0.75 milliliters per minute for pump two. Set the volume to any value above five milliliters and select the infusion mode for both pumps.

Then click set to confirm the settings. To prepare an LTSL10 or LTSL10 ICG lipid mixture, use one five milliliter luer lock syringe to withdraw one milliliter of lipid mixture and another five milliliter luer lock syringe to withdraw at least three milliliters of ammonium sulfate solution. Slide the barrel flange of the syringe to the syringe retainer of the pump to install the loaded syringes onto the syringe pumps in the upright position and attach the plunger flange of the syringe to the pusher block of the pump and wrap the other end of the heating tape and the temperature probe with the thermostat around the syringe containing the lipid solution.

Wrap the end of the heating tape to the syringe containing the aqueous solution, connect the syringe containing the lipid mixture to the ethanol inlet, and connect the syringe containing the ammonium sulfate solution to the aqueous inlet. Adjust the plunger position to remove any air bubbles from the syringes as necessary and plug and unplug the heating tape in 10 second intervals until the temperature of the syringes reaches about 51 degrees. When the thermostat shows an appropriate temperature, click run all in the pump control software to run the syringe pumps and check that the fluid flow is free of air bubbles and any leakage.

Collect the liposome samples into a vial and pause or stop the infusion when either syringe is almost empty. Anneal the collected liposome solutions in a 60 degree Celsius water bath for 1-1/2 hours before transferring the solutions to dialysis tubes for dialysis against one liter of 240 millimolar ammonium sulfate for at least four hours at 37 degrees Celsius. Then store the purified liposomes at four degrees Celsius.

For remote loading of the liposomes by transmembrane pH gradient, load 25 milliliters of HBS onto the top of a size exclusion chromatography column and allow all of the eluent to flow through the column. Load one milliliter of dialyzed liposomes to the column. When all of the lipid solution has passed through the column, add 1.5 milliliters of fresh HBS to the column.

When all of the buffer has passed through the column, add three milliliters of fresh HBS to the column and collect the eluent. Next, add DOX solution at a 1:20 DOX:phospholipid molar ratio to one milliliter of buffer exchange liposome solution in a Biju vial and place the vial in a 37 degree Celsius water bath for 1-1/2 hours. After loading, purify the liposomes by size exclusion chromatography as just demonstrated.

For laser heating induced trigger release of the liposome contents, set the water bath to 37 degrees Celsius. When the temperature has stabilized, add 200 microliters of DOX loaded liposomes to each well of a clear 96-well plate and place the plate in the water bath with the bottom submerged in the water. Then set the laser system current to 2.27 amps and place the laser system collimator five centimeters vertically above the surface of the 96-well plate.

Switch on the laser and use a fiber optic temperature probe to monitor the temperature once a minute. At five and 10 minutes, aspirate 10 microliters of DOX loaded liposomes from each well of the clear 96-well plate and add 190 microliters of HBS to three individual wells of a black 96-well plate. To assess complete drug release, mix 10 microliters of the liposomes with 170 microliters of HBS and 20 microliters of 1%Triton X-100 detergent solution in three individual wells of the black 96-well plate.

Then measure the DOX fluorescence intensity on a plate reader. Microfluidic production of LTSL4 results in a gel-like viscous solution illustrated by the large number of trapped air bubbles while the preparation of LTSL10 results in the formation of a clear non-viscous liquid. Dynamic light scattering measurement of LTSL10 prepared at 51 degrees Celsius demonstrates the expected Z-average diameter and dispersity indicating the success of the experiment.

When LTSL10 is prepared at 20 degrees Celsius, however, larger and more dispersed liposomes are obtained resulting in a suboptimal product. LTSLs prepared by the conventional method of lipid film hydration with extrusion demonstrate a DOX encapsulation efficiency of 50-80%With annealing, LTSL10 prepared by microfluidics results in a significant increase in DOX encapsulation efficiency to a mean of 85%indicating the success of the remote loading of DOX and the presence of the transmembrane pH gradient. The DOX release profile of LTSL10 has been determined to be temperature sensitive.

LTSL10 has a relatively broad phase transition with onset at 41.6 degrees Celsius that peaks at 42.6 degrees Celsius. The efficiency of the ICG loading is dependent on the initial ICG concentration and the size and dispersity of the samples. Further, LTSL10 ICG irradiation with a near infrared laser induces photothermal heating triggering the release of DOX.

When attempting this procedure, it is important to ensure a stable fluid flow such that the mixing of fluid and therefore the formation of liposomes remain reproducible. Liposome annealing and dialysis are two important steps for ensuring a stable liposome formulation with high loading capacity. In vivo testing can also be carried out to assess LTSL10 bio-distribution, drug release, and anticancer activity.

Our protocol can be used for the successful microfluidic production of cholesterol-free lysolipid-containing thermosensitive liposomes for drug delivery applications.