Appendix A

Appendix A: A Standard Procedure to Produce SE61

 

1. To a clean beaker, add 1.5g of sodium chloride, 1.464 g of Span 60, and 1.288 g of TPGS in 50mL of PBS, and a stirrer bar.

• This will result in 50 mL of solution, can multiply each ingredient by 2 to get 100 mL, and so on.
• The smaller the pieces of TPGS, the less time for it to melt

 

2. Cover this beaker with aluminum foil and put a piece of “autoclave” tape on the foil.

 

3. Heat/stir this beaker (not too hot) until you see it begin boiling. 

 

4. Let the solution boil until Span and TPGS completely melt/dissolve, approximately 1-2 hours.

 

5. Autoclave for approximately 35 min. This reduces Span60 particle size.  ***

 

-Possible to stop here, put mixture in 4°C fridge, and continue another day-

 

6. While the mixture is cooling, it would be a good idea to put separation funnels (needed later on) in the fridge so that they will be cold. 

• Use the 250 mL separation funnel, better separation/results
• Need 50 mL per funnel, so use as many as you want/need

 

7. After the mixture has cooled to room temperature, prepare for sonication.  (MAKE SURE TO REMOVE MAGNETIC STIRRER BAR)

• Transfer 50 mL of solution to a 300 mL (tall) beaker and set up in ice bath

 

8. Purge the mixture with PFC gas until bubbles cover the solution before sonication (about 15 s @ 15 ml/min).

• NOTE: When purging with gas, the tip that supplies the gas WILL be in the solution.  When sonicating, the tip supplying the gas will NOT be in the solution, only the sonication probe will be submerged.  (Make sure to wear protective headphones)

 

9.  The solution is sonicated (Misonix Inc. CL4 tapped horn probe with 0.5” tip, Farmingdale, NY) at 110W for 3 minutes in ice bath under a steady stream of PFC gas. 

 

10.  When sonication is complete, remove the beaker and retrieve the cold separation funnels from the fridge.  Empty the contents of the beaker into one of the separation funnels (make sure the bottom is closed). 

 

11. Wash the beaker with 20mL cold membrane-filtered PBS and add 30mL of cold PBS to the separation funnel. Place it in the fridge for 1.5-2 hours

 

• Separation becomes clear after an extensive period of time, sometimes it is a little more than 1 hour, usually 1.5-2 hours. Easiest to see demarcation with 250 mL separation funnel, check occasionally
   
  

Figure 1 - Separation funnels with SE61 (loaded with Nile Red), can clearly see separation.

Removing bottom layer in left funnel

12. After the allotted time, remove the separation funnel from the fridge and discard the bottom layer (when using 50 mL of original solution, discard volume ranges 40-70 mL). Add another 50 mL of cold PBS to the funnel and replace in fridge. Time separation, same as before (maybe a little less).

 

13. After the allotted time, repeat 13.

 

14. After the allotted time (4 separations total), collect the bottom layer

 

15. Collect the second layer in increments of 5mL in separate containers

 

16. Take 1mL of solution from each 5mL container for analysis under a microscope.  This will be used to determine whether the bubbles are too large.  If they are too large (greater than 3 microns), discard them.

 

17. Pipet 1 mL in lyophilization vials with 0.5 mL 400 mM glucose lyoprotectant and 0.5 mL plasma-treated PBS to prepare for freeze-drying.

 

• Working solution is 1:1 microbubbles in membrane-filtered PBS

 

 

*** Autoclave:  Take out the inner part of the autoclave and measure the water level.  The water level should be between the 2-2.5 inch mark, if not, adjust the water level accordingly.  Turn the heat to maximum so that the water begins heating.  After putting in the beakers, put the top back on the autoclave and screw all the knobs in place. Wait until seeing the water boil then close a ventilation valve. Wait until the pressure inside reaches the green zone, adjust the heat to 4, and count 35 minutes from there.  Make sure to release pressure accordingly using the control valve.  Also, be sure to wear the mitts to avoid causing injury.  

 Appendix D