Liposome Nanoparticles

Safety Procedures

Please be aware of any potential hazards prior to operating any equipment. Both cholesterol and DLPC solutions are produced using chloroform and will be provided for you start the experiment. If not provided, please contact TA or staff. Keep all chloroform solutions inside the fume hood and avoid inhaling any fumes produced in the experiment. In the event of exposure, run water over the affected area, contact lab personnel, and if additional medical attention is required use the protocols at the lab exits to contact EH&S or call 911. In event of fume inhalation,  ask a lab member to assist you in moving to the nearest exit, contact lab personal, and use the protocols in the Emergency Response Guide at the lab exits to contact EH&S or call 911.

Other Safety Concerns:

• Wear a lab coat, gloves, and safety glasses at all times.

• Only work in the fume hood.

• Do NOT pour waste down the drain! A labeled waste bottle should be in the hood for all waste; if you can't find it then notify an instructor or TA and you'll be provided one.

• Be careful when handling syringes and needles for the extrusion procedure and make a note of nearby first aid kits in case of needle puncture.

• Dispose of broken glassware in the appropriately labeled receptacles; used filters and spacers can be disposed of in the trash.

Programs, Equipment, and Instruments

• Extruder housing with two Teflon blocks

• Two 1 mL syringes

• Sonicator

• Vortex mixer

• DLS with particle sizing software

Objectives

This is more like a wet-chemistry lab than an engineering experiment. Your goal is to make 100 nm liposomes and characterize them using dynamic light scattering (DLS), and the recipe to do so is as follows:

1. Create a lipid solution.

   1. Pipette 450 µL phospholipid stock, 50 µL cholesterol stock, and 1.5 mL chloroform into glass vial.

   2. Cap and gently shake to combine.

2. Hydrate a lipid film.

   1. Use an air stream to gently evaporate all chloroform from the lipid solution to yield a lipid film on the vial walls.

   2. Hydrate the lipid film by adding 2 mL deionized water.

   3. Agitate the lipid film first using the vortex mixer (~30 s), then using the sonicator (~2 min).

3. STOP: Before extrusions, ask a TA to receive proper training on the extruder assembly!

4. Extrude your sample. 

   1. Disassemble the extruder, then add spacer/membrane/spacer (in that order) to one side of the Teflon block. Reassemble the extruder, taking care to tighten the nuts enough to prevent leaks but not rupture the membrane.

   2. Load your sonicated solution into a 1 mL glass syringe.

   3. Carefully twist the loaded syringe onto the one of the luer-lok connections of the extruder assembly. Connect the empty syringe onto the opposite side.

   4. Push the syringe plunger slowly to perform an extrusion. The extrusion is complete when the plunger is fully depressed. At this time the empty syringe should be mostly filled and its plunger should be extended.

   5. Push the plunger of the now-filled syringe to perform another extrusion. Repeat this process for the desired number of extrusions (usually 5-15).

5. STOP: Before performing DLS analysis you must receive training.

6. Characterize with DLS. 

   1. Transfer your sample to a cuvette. If the liquid level is below the fill line, add deionized water until the liquid level reaches the fill line.

   2. Cap your sample.

   3. Place your sample in the DLS instrument. The laser beam path is from left to right, so orient your cuvette appropriately.

   4. Run the analysis using the instruction card near the computer.

7. Clean up.

   1. Disassemble the extruder and syringes. Rinse both with DI Water and set in the fume hood to dry.

   2. Collect all waste in the waste bottle in the fume hood.

   3. Dispose of membranes, spacers, and pipette tips in the lab trash.

   4. Check with a TA if you have any extra solutions that need to be stored.

Tips

1. This is one of the only labs where there is a "right" answer: you're to produce nanoparticles that are nano-sized (micron-sized particles, for example, may result in a point deduction).

2. A common variation in the lab is to vary the composition of the lipid solution or the number of extrusions.

3. The number of extrusions should always be odd.

4. Your team should attempt to make no more than about 3-5 samples per day.

5. Your discussion should address (at minimum) any unusual results and means to improve your sample. Be sure to explain why your suggested improvements are expected to improve the samples, with specific reference to what happens to the lipid film/particle during the extrusion process.

6. You can see an overview of how DLS works in the video below.

References

1. Zhang, L.; Granick, S. Nano Lett., 2006, 6, 694-8.

2. Zhang, L. et al. Clin. Pharmacol. Ther., 2008, 5, 761-9.

3. Torchilin, V.P. Nat. Rev. Drug Discov., 2005, 4, 145-160.

4. Barenholz, Y. Curr. Opin. Coll. Int. Sci. 2001, 6, 66-77.

5. Mayer, L.D.; Hope, M.J.; Cullis, P.R. Biochim. Biophys. Acta, 1986, 858, 161-168.

6. Yang, D.R. et al. Biomaterials, 2009, 30, 6035-6040.