Investigating Lipids In today s culture, there is a stigma associated with the word fat. While it is true that too much fat can lead to health problems, fats (or lipids) play very important roles in biology. In fact, none of us would be here without them. As a macromolecular group, lipids are involved in the formation of membranes and steroid hormones, the storage of potential energy, protecting organs and stabilizing temperatures, all of which help to maintain homeostasis. Of course, lipids can only carry out these important jobs due to their chemistry and chemical structure. Most biological lipids are hydrophobic, due to their abundance of carbon and hydrogen atoms. Some lipids are modified to contain additional hydrophilic groups, making them amphipathic (molecules that contain both hydrophilic and hydrophobic moieties within the same structure). Lipids can be a challenge to work with because of the significant hydrophobic effect that naturally draws them together in an aqueous environment. To this end, most lipid investigations require the use of organic solvents, such as chloroform, ether, or alcohols to solubilize them. In today s lab, we will investigate some of the properties of the lipids. We will look at the ability of lipids to help insulate against thermal changes and their natural tendency to form aggregates and membranes in an aqueous environment. Briefly, we will be comparing the insulating ability of a wax, a saturated fat, an unsaturated fat, a hydrogenated oil, and water to insulate a volume of water. The other exercise is to create liposomes and test their ability to encapsulate and shuttle molecules into cells. The Insulating Ability of Lipids: THIS IS A TIMED EXPERIMENT.MAKE SURE YOU WORK LIKE WELL OILED MACHINES TO OBTAIN THE BEST RESULTS!! 1. Each group should obtain a digital thermometer and a digital timer from the table and become familiar with their working. 2. Each group must retrieve from the 37 degree incubator oven the following: a set of cans containing 37 degree wax, 37 degree lard, 37 degree oil, 37 degree hydrogenated oil (crisco), and 37 degree water. The cans contain the same volume of insulator, be careful not to spill the can with water and oil. 3. Each group will also have to quickly obtain a container of 37 degree water from the incubator. This volume of water will be quickly and carefully pipetted into the central tube inside each of the can. 4. Once back at your table, quickly and carefully transfer 25 ml of the water into each of the central tubes jacketed by the different insulating agents. 5. Immediately obtain the initial temperature of the water sample and record it. Try to record the temperature that tends to stabilize on the thermometer probe. 6. Start the timer counting up. Every 5 minutes, record the temperatures of the water samples in the tubes in each of the cans. 7. Obtain temperature data points over an hour time period, by filling in the chart on the following page. 8. When done, dump the water from the inner blue cup and return the set ups to the 37 degree incubator.
Time, Minutes 0 Wax Lard Oil Crisco Water 5 10 15 20 25 30 35 40 45 50 55 60 9. In a graphing program, such as prism or excel, generate an XY graph, plotting temperature on the Y axis vs. Time on the X axis for each of the samples. 10. Analyze each data set using linear regression to obtain the slope of the data points. Remember that slope is the change in Y over the change in X. In our case the change in temperature vs the change in time. This value will be the rate of cooling for each insulator and will allow you to compare them to each other. 11. Observe and record your results. The Formation of Liposomes and Their Interaction with Cells: Phospholipids and other amphipathic molecules will spontaneously aggregate in an aqueous environment, driven mainly by entropic effects and hydrophobic interactions. This phenomenon, of course, is responsible for cellular membranes and organelle structures. Liposomes are hollow spheres of membrane lipids which can encapsulate a variety of water soluble materials. Studies using liposomes have revealed their usefulness as delivery vehicles for materials into cells. When a liposome, bearing some kind of soluble cargo on the inside, encounters a cell s membrane, they both fuse together
and the soluble cargo material is transferred into the cell. While the production of liposomes is technically easy (sonicating membrane lipids and cargo in an aqueous environment), the formulation of their composition can be challenging. Different ratios of membrane components combine to create liposomes with unique stabilities. Some recipes work better than others. In this exercise, your group will develop a few different recipes of your own to find out which one creates the most stable liposomes. Then you will attempt to use your formula to create fluorescent liposomes, lipsomes carrying a dye called fluorescein. Finally, your fluorescent liposomes will be applied to live mammalian cells to determine whether your liposomes can transfer their fluorescent cargo into the cells, making them fluorescent. We will use the fluorescent microscope to determine this. Keep track of your recipes...you may develop a winner! The common amphipathic membrane lipids you will be using are illustrated and named below. They include: 1. Phosphatidylcholine, 2. Phosphatidylenthanolamine, and 3. Cholesterol. The cargo for the liposomes includes trypan blue and fluorescein.. Phosphatidylcholine 1,2-Dioleoyl-Glycero-3- Phosphoethanolamine (DOPE) cholesterol liposome
The structure of trypan blue The sodium salt of fluorescein Each group will also have to obtain a light microscope from the shelves. 1. In the fume hood, using GLASS test tubes and the lipid reagent solutions. Create and combine different lipsome lipid formulations. The lipids are in a chloroform solvent so measuring them using plastic micropipettors and tips is not useful. Instead, use drop number from a glass pasteur pippette to keep track of your recipes. Make as many combinations as you like, but do at least four. Example: 1 drop phosphatidylcholine + 2 drops phosphatidylethanolamine + 1 drop cholesterol. 2. The heat of your hand on the tube and the volatility of the chloroform should allow your lipid mixture to evaporate quickly, leaving a film on the inner surface of the tube. Once completely dry, you are ready to add your payload. 3. load your liposomes first with trypan blue. This is a visible blue dye that will create liposomes visible with he standard light micrscope. To load your lipsomes, add 1-2 drops (keep track) of the trypan blue solution (it is dissolved in an aqueous PBS buffer) and place in the sonicator for 30 seconds to 1 minute (Keep track). 4. After sonication, transfer some of your mixture onto a microscope slide and add a coverslip. Observe and rate your formula for effective liposome production. Get your partner s opinion as well. 5. When you are satisfied with your best recipe, recreate it, but this time use the fluorescein/pbs buffer solution, instead of trypan blue. 6. When your are ready, see Dr. T. for a chamber slide of live mammalian cells. 7. Note that you have several wells of live cells on the chamber slide. The wells of cells are to test your liposomes as well as running a staining control. 8. Carefully remove the cell media from each well you will test and carefully rinse each well the same way using ice-cold PBS buffer. BECAREFUL NOT TO DISLODGE TOO MANY CELLS GROWING ON THE BOTTOM OF THE SLIDE!!!
9. Into one of the wells of cells, add a drop of your fluorescein liposomes. Dilute the well with some PBS buffer to ensure all of the cells are covered. Keep track of the drop number of fluorescein liposomes and PBS added the well. 10. Into another well, add the same drop number of fluorescein buffer (used before the liposomes were made) and PBS buffer. 11. Into a third well, add the same drop number of PBS, but No fluorescein. 12. Incubate the slide with cells on ICE for 10 minutes. 13. After the incubation period, carefully remove the contents of the wells and carefully rinse each well the same way using cold PBS buffer. Repeat three times. 14. After the final rinse, carefully remove the plastic cambers from the slide, add coverslips to the wells you used, and see Dr. T. for help using the fluorescent microscope. 15. Record your data and make obervations to determine how successful you were! Questions: 1. Print out a nice looking graph of your temperature experiment data 2. From your data, which insulating agent was the most effective? Back up your answer with a logical interpretation of your data. 3. From your data, which insulating agent was the least effective? Back up your answer with a logical interpretation of your data. 4. Do saturated or unsaturated fats insulate better? 5. After observing your data, write down a hypothesis about what makes a material a good insulator. 6. Which liposome formula gave the best results? 7. Looking at the structures of typan blue and fluorescein, explain which specific functional groups and/or bonds contribute to their hydrophilicity. 8. Include fluorescent micrographs of your liposome experiment. Were you able to load a live cell with fluorescent cargo?