A Technique to Generate a Novel Delivery Platform for a Delayed Burst Release of Molecules
Transkript
To fabricate the polybubbles, first, use a 1-milliliter transfer pipette to add 800 microliters of 10% CMC into a 0.92-milliliter glass vial. To synthesize PCLTA, mix 1,000 grams per milliliter of 14 kilodalton PCL in 200 microliters of DCM. To synthesize PLGADA, mix 1,000 grams per milliliter of 5 kilodalton PLGADA into 200 microliters of chloroform.
Mix photoinitiator with the polymer mixture of interest at a 0.005-to-1 ratio and load 200 microliters of the resulting solution into a 1-milliliter glass syringe mounted on a syringe pump connected to a dispensing stainless steel tube with an inner diameter of 0.016-inch.
Using a micro-motor to control the forward and backward motion of the polymer tube, inject the polymer into the 10% CMC in the glass vial to form the polybubbles and cure the polybubbles under ultraviolet light at a 254-nanometer wavelength for 60 seconds at 2 watts per square centimeter. Then, flash-freeze the polybubbles in liquid nitrogen for 30 seconds and lyophilize the carriers overnight at 0.01 millibar vacuum and minus 85 degrees Celsius.
For centering of the cargo of interest within a polybubble, mix the cargo with 5% CMC and a rotator overnight to increase the viscosity of the cargo before manually injecting 2 microliters of the cargo mixture into the poly bubble. When all of the cargo has been injected, recure, flash-freeze, and lyophilize the injected polybubble as just demonstrated.
The next morning, use forceps to separate the polybubble from the dried CMC and wash the polybubble with deionized water to remove any residual CMC. Then, cut the polybubble in half and image the halves by confocal microscopy to ensure that the cargo is centered.
For small-molecule cargo release, incubate the polybubbles with centered acriflavine and 400 microliters of PBS at 37 degrees Celsius for the appropriate incubation length. At each experimental time point, collect the supernatant, and add 400 microliters of fresh PBS to the vial. Then, use a plate reader to quantify the fluorescence intensity of the collected supernatant.
