This protocol can help maintain the cargo stability, which can be both small molecules and proteins, through the use of the polybubble, which can in turn reduce the carbon interactions with the solvent. This novel technique can be used to improve vaccine coverage in the developing countries as vaccine functionalities can often be compromised due to inefficient transportation and storage facilities. To fabricate the polybubbles, first use a one 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 five 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 one 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 two 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 in minus 85 degrees Celsius. For centering of the cargo of interest within a polybubble, mix the cargo with 5%CMC in a rotator overnight to increase the viscosity of the cargo before manually injecting two microliters of the cargo mixture into the polybubble. When all of the cargo has been injected, re-cure, 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 in 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. For near-infrared activation, mix the polymer solution with hydrophobicized gold nano rods at a one to nine ratio and add photo initiator at a 0.005 to 1 ratio.
Inject the resulting mixture into a 0.92 milliliter glass vial containing 800 microliters of 10%CMC and cure the polybubbles under ultraviolet light as demonstrated. After flash freezing and lyophilization as demonstrated, wash the polybubbles with deionized water and incubate the carriers in 400 microliters of PBS at 37 degrees Celsius for the appropriate incubation period. At the end of the incubation, obtain a forward looking infrared image of the polybubble before and after activating the polybubbles with an 801 nanometer near-infrared laser at eight amps for five minutes three times a week for four weeks for PLGADA polybubbles and 14 weeks for PCL-PCLTA polybubbles.
At each experimental time point, collect the supernatant and add 400 microliters of fresh PBS to the vial. Then calculate the temperature differences between the polybubbles before and after laser activation based on the temperature values from the forward looking infrared images. The addition of 10%CMC-based aqueous solution results in a complete polybubble suspension for a successful maintenance of the polybubble sphericity.
Cargo injection into the polybubble in the absence of CMC results in leakage and a consequent lack of cargo retention within the polybubble. To address this leakage, the viscosity of the PCLTA can be increased using potassium carbonate isolated after end capping polycaprolactone with triacrylate and the viscosity of the cargo can be increased by mixing the cargo with 5%CMC. The viscosity of the PLGADA polybubbles is sufficient to facilitate centering of the cargo and does not require modulation via potassium carbonate.
Note that no statistically significant difference in the binding efficiency of the antibody is observed after mixing HIV gp120/41 antigen with and without trehalose before polybubble injection. Delayed burst releases are observed in PLGADA polybubbles with acriflavine in the middle on day 19 for polybubbles incubated at 37 degrees Celsius and on day five for polybubbles incubated at 50 degrees Celsius. In addition, PLGADA polybubbles and PCL and PCLTA polybubbles containing gold nano rods can be successfully laser-activated multiple times.
When forming the polybubbles, please take care to protect the reagents from the light and also to use the solutions immediately. After developing this technology, we also wanted to explore automating this process to the scale up the polybubble production.
