Nanoparticle shape influences cellular uptake, but limited work has been done to change the shape of polymersomes. This protocol demonstrates how to elongate polymersomes for enhanced drug delivery applications. This technique provides a simple approach that can create many different polymersome shapes with potential applications in drug delivery, pseudo cells, or for other unexplored fields.
Elongated polymersomes can simultaneously deliver hydrophobic and hydrophilic drugs and more easily in to the cells. This technique could help shuttle drugs across the blood-brain barrier, addressing a major medical challenge. To begin, dissolve 0.015 grams of selected polyester in one milliliter of DMSO by vortexing for 15 minutes, and set up the solvent injection apparatus.
Place the start plate directly below the vertical syringe pump, then place a five milliliter glass bile with one milliliter of type two deionized water in a miniature star bar on the stir plate. Adjust the syringe pumps height to allow for the tip of the needle to be fully immersed in type two deionized water and set the infusion rate of the syringe pump to five microliters per minute. Perform the solvent injection by drawing the organic solvent and polyester solution into a 27 gauge needle.
Place the needle into the syringe pump and make sure that it is secure. Adjust the pusher block to touch the syringe plunger's end. Start the stir plate so that the water is spinning at 100 revolutions per minute, and then start the syringe pump.
Once the syringe pump has fully infused the organic solvent and polymer into the water, remove the stir bar and cap the glass vile. For characterization of the polymersome via dynamic light scattering or DLS, add one milliliter of water with a small percentage of organic solvent and polymer to a one milliliter cuvette. Performed DLS by placing the cuvette into the system and setting up the run.
Read the polymersome intensity weighted diameter and polydispersity index or PDI. After washing a 300 kilodalton dialysis membrane according to protocols provided by the manufacturer, add one milliliter of polymersome solution into the reservoir of the dialysis device. Place the dialysis device in the 250 milliliter beaker with 150 milliliters of type two deionized water on a stir plate.
Set the stir plate to a speed that allows for gentle movement of the dialysis device and leave it to stir overnight. After the dialysis has completed, extract the one milliliter polymersome solution from the dialysis device. Create 150 milliliters of desired salt buffer with either 50, 100 or 200 millimolar concentration of sodium chloride based on the final desired polymersome properties and keep the load of dialysis device in 150 milliliters of salt solution for 18 hours for stirring.
After shape modulation, perform DLS measurements. Pay particular attention to PDI measurements compared to spiritual polymersomes as a change in PDI suggests an effective shape change in polymersomes. Use appropriate controls for imaging, especially non-shape modulated polymersomes to ensure the method's success.
TEM observation of PEG-PLA based polymersomes indicate an overall spherical structure with a thicker exterior line that is indicative of a membrane. The polymersomes present as physical structures and SEM with a brush like exterior layer of PEG. One hour dialysis in water led to the same overall average diameter with solvent removal decreasing the polymersome diameter.
When larger initial concentrations of organic solvent are used, larger diameter decreases are expected. While making the PEG-PLA polymersomes, modest changes in PDI are expected, which may indicate a change in shape. When dialyzing PEG-PLGA polymersomes, which are slightly more hydrophobic than PEG-PLA polymers against salt, the increase in PDI is more consistent with elongation with all explored salt gradients leading to an increase in PDI.
Similar results should be observed when using a 50 millimolar salt gradient to cause a shape change regardless of polyester hydrophobicity. Meanwhile, 100 and 200 millimolar sodium chloride salt gradients display the direct trend that delta PDI increases with increasing polyester hydrophobicity. TEM images of PEG-PLA polymersomes dialyzed with 50 million lower sodium chloride showed with stomatocytes and elongated rods.
As salt concentration increased to 100 millimolar, an increased number of rods formation with a decreased number of stomatocytes was observed. Dialysis against 200 millimolar polymersomes more consistently formed prolates with modest aspect ratios. The most challenging part of this protocol is making the polymersome consistently.
So it is important to practice the procedure.
