User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/06/25/Liposomes and kinesin

Calculating the amount of liposomes needed

The first thing I want to do is to calculate the amount of liposomes we will be using to coat each chamber. To do this I will start with the surface area for a lipid from a paper that I will need to find the reference for.

[math]\displaystyle{ 0.4 \mathrm{\ } \frac{\mathrm{nm}^2}{\mathrm{Lipid}} }[/math]

I have 100 nm filters that will make (nominally) 100 nm diameter liposomes. The number of lipid molecules per liposomes is

[math]\displaystyle{ \begin{align} \mathrm{Surface\ area\ of\ a\ liposome} = 4\pi r^2&\approx 31416\mathrm{\ nm}^2\\ \mathrm{Surface\ area\ of\ the\ bilayer\ of\ a\ liposome} = 2\times 4\pi r^2 &= 62832\mathrm{\ nm}^2\\ \frac{\mathrm{Lipid}}{\mathrm{Liposome}} = \frac{62832}{0.4} &= 157080\mathrm{\ }\frac{\mathrm{Lipids}} {\mathrm{Liposome}} \end{align} }[/math]

Using the average molecular weight of L-α-phosphatidylcholine of 770 g/mole, I can find the number of liposomes I can make with the 25 mg of lipid molecules I have.

[math]\displaystyle{ \begin{align} \left(770\mathrm{\ }\frac{\mathrm{g}}{\mathrm{mole}}\right)^{-1}\mathrm{\ }6.022\times10^{23}\mathrm{\ }\frac{\mathrm{Lipids}}{\mathrm{mole}} &= 7.8\times 10^{20}\mathrm{\ }\frac{\mathrm{Lipids}}{\mathrm{g}}\\ 7.8\times 10^{20}\mathrm{\ }\frac{\mathrm{Lipids}}{\mathrm{g}}\mathrm{\ }25\times 10^{-3}\mathrm{g} &= 2\times10^{19}\mathrm{\ Lipids}\\ \mathrm{\#\ of\ Liposomes} &= 1.2\times10^{14}\mathrm{\ Liposomes} \end{align} }[/math]

The next thing I want to find is the amount of liposome solution required to fully coat the surface area of our chamber. We are using cover slips and cover glass slides that are 24 mm and 25 mm in width respectfully.

[math]\displaystyle{ \begin{align} \mathrm{Surface\ area\ of\ chamber} &= 600\times10^{12}\mathrm{\ nm}^2\\ 1.2\times 10^{14}\mathrm{\ Liposomes} / 2.5 \mathrm{\ mL} &= 48\times10^{12}\mathrm{\ }\frac{\mathrm{Liposomes}}{\mathrm{mL}}\\ 600\times 10^{12}\mathrm{\ }\frac{\mathrm{nm}^2}{\mathrm{Sample\ chamber}}\mathrm{\ }\frac{\mathrm{Liposome}}{31416\mathrm{nm}^2} &= 1.9\times 10^{10}\mathrm{\ }\frac{\mathrm{Liposomes}}{\mathrm{Chamber}}\\ \frac{1.9\times 10^9\mathrm{\ }\frac{\mathrm{Liposomes}}{\mathrm{Chamber}}}{48\times 10^{12}\mathrm{\ }\frac{\mathrm{Liposomes}}{\mathrm{mL}}} &= 0.4\mathrm{\ }\frac{\mathrm{mL}}{\mathrm{Chamber}} \end{align} }[/math]

Thus the amount of liposome sauce needed to coat our sample chambers is

[math]\displaystyle{ \begin{align} 400\mu\mathrm{L} \end{align} }[/math]

Update

After making the above calculation, I realized that 400 µL may be too much liquid for the volume in the sample chamber. Estimating the thickness of the Scotch tape to be approximately 50 µm, the sample volume has about 300 µL 30 µL of volume in it. This means I need to recalculate for 300 µL 30 µL being the amount of liposome sauce I want to add. Since the liposome solution was toasted by the aspirator, I'll redo this calculation when we get new lipids.

Making liposomes

The lipid molecules come packed in chloroform. We need to get rid of this chloroform by using my patented SpinMaster Micro™.

This device takes the place of a much more expensive rotary evaporator. The idea is to blow nitrogen into the vial that has the lipid molecules and spin it. This evaporates the chloroform and leaves a nice uniform coating of lipid molecules in the vial. From experience, it is much easier to get lipid molecules off of glass when they are in a uniform film as opposed to clumps. Clumps happen when you try to evaporate the chloroform by hand rotating the vial.

The next step ideally is to put the liposomes under vacuum for a few hours to remove the rest of the chloroform. Right now I'm struggling to setup the aspirator to do this because we do not have a vacuum pump.

I finally got it to work. However, I'm wasting a lot of water to do so. Typically one should keep the dried lipids under vacuum for at least 2 hours but I'm not going to have it go for that long.

I will never use an aspirator again! Well, the water went into the vacuum chamber through the aspirator and ruined the lipids. Yeah.