The overall goal of this nanosponge synthesis technique is to understand and utilize important synthesis parameters for design and control of size in morphology. This method will help you to produce nano particles in precise dimensions and crosslinking densities for drug delivery applications. The main advantage of this technique is that it can be tailored towards different crosslinking densities of these particles and also towards different sizes of these particles and this is a very important factor in many applications where these particles will be used.

Though this method can provide insight into nanoparticle design through utilizing tailored wool synthesis parameters, it can also be applied to other polymer networks such as hydrogels and microparticles. Remove moisture from a 25 milliliter round bottom flask equipped with a magnetic stir bar as detailed in the text protocol. Once cooled, quickly remove the septum from the round bottom flask and add 2.5 milligrams of tin triflate to the very bottom of the flask, using a spatula.

Replace the septum. Sequentially, add 72.6 microliters of 3-methyl-1-butanol via a 100 microliter microsyringe and 1.33 milliliters of anhydrous DCM via a two milliliter syringe. Stir the suspension on a magnetic stir plate for 10 minutes.

Next, sequentially add 0.48 milliliters of AVL and 1.37 milliliters of VL.Allow the reaction to continue stirring for 18 to 20 hours. Quench the reaction by adding approximately five milliliters of methanol, then, add 100 milligrams of solid support kin scavenger to the flask and stir for two hours. Filter the suspension by gravity filtration to remove the solid, then, rinse the flask with dichloromethane solvent and pour this over the filter.

Remove the solvent under rotary evaporation, with a water aspirator as the vacuum source, while heating at 30 degrees celsius until the solution is viscous. Precipitate the solvent drop wise, into 500 milliliters of cold methanol to produce flakes of white solid. Then, filter the solution by vacuum filtration into a funnel containing a fretted glass disk with filter paper to collect the solid.

Transfer the solid product to a pre-weighed vial via a spatula and dry overnight using a high vacuum pressure of 05 Torr to collect the white flaky solid. Add 500 milligrams of the VLAVL co-polymer to a six stream vial with a magnetic stir bar. Then, add 6.15 milliliters of anhydrous DCM to the vial and vortex to solubilize the polymer.

Next, add 74.53 milligrams of mCPBA to a second six stream vial. Add 6.15 milliliters of anhydrous DCM and vortex until the mCPBA is completely solubilized. Next, transfer the mCPBA solution to the VLAVL solution.

Cap the reaction and cover with a plastic paraffin film, before allowing it to stir for 48 hours. Transfer the reaction mixture to a 50 milliliter separatory funnel and add 15 milliliters of saturated sodium bicarbonate. Cap the separatory funnel and rock gently to mix.

Collect the organic layer containing the product and transfer it into a 15 milliliter Erlenmeyer flask with an appropriate size magnetic stir bar. Add five milliliters of DCM to the aqueous layer that is still in the separtory funnel, cap and then gently rock. Collect the organics and transfer them into the product flask.

After discarding the aqueous waste, transfer the organic layer back into a separatory funnel. Next, add magnesium sulfate to the product flask while stirring over a magnetic stir plate to remove any residual water, continue adding small scoops of magnesium sulfate until it no longer clumps when added. Use a glass funnel fitted with a filter paper to remove the solid magnesium sulfate while transferring the mixture to a 50 milliliter round bottom flask.

Transfer the contents of the round bottom flask to a pre-weighed product vial. Remove the solvent by rotary evaporation with the water aspirator as the vacuum source and heating at 25 degree celsius. Place the vial on high vacuum at 05 Torr overnight to produce a white waxy solid.

Dissolve 200 milligrams of the VLAVL EVL polymer, in 20.01 milliliters of anhydrous DCM, for an apoxide concentration of 0054 molar. Transfer the resulting solution to a 100 milliliter round bottom flask, with a 1420 neck. Place the reaction flask in an oil bath at 50 degree celsius.

Stir the solution with a fast vortex and add 21.45 microliters of EDEA drop wise via a microsyringe. Fit the neck of the flask with the water jacketed condenser that has cool water flowing through it, fitted with a 1420 neck adapter and reflux the solution for 12 hours. Next, remove excess solvent from the reaction flask by rotary evaporation at 25 degrees celsius until a viscous solution is obtained.

Transfer the product to 10k molecular weight cutoff dialysis tubing that has one end folded and closed with the dialysis clip. Rinse the flask with excess DCM and transfer it to the tubing. Fold the top of the tubing and close it with a dialysis clip that has a wire for hanging.

Then, hang the dialysis tubing on the side of a two liter beaker with a large stir bar and fill the beaker with DCM until the dialysis tubing is completely submerged. Gently stir the dialysis solution on a magnetic stir plate and cover the beaker with aluminum foil to prevent solvent evaporation. Remove the solvent by pouring into a waste container and replace with fresh DCM three times daily, for 48 hours, to remove the unreacted polymer and crosslinker.

Following dialysis, remove all solvent from the beaker and transfer the contents of the dialysis tubing to a 10 milliliter syringe fitted with a 0.45 micron PTFE syringe filter. Push the solution through the filter directly into a pre-weighed product vial to remove any remaining impurities. Remove the solvent by rotary evaporation at 25 degree celsius, then, place the product vial on high vacuum at 05 Torr overnight, to collect a light yellow waxy solid.

Place 0.5 milligrams of nanosponges into a 1.5 milliliter centrifuge tube. Add one milliliter of filtered cell culture water. Use a probe sonicator to sonicate the solution with two second bursts, four to five times at room temperature, until the particles have developed a fine suspension.

Then, add 30 milligrams of PTA to one milliliter of filtered cell culture water in a 1.5 milliliter centrifuge tube. Vortex on the highest setting for 10 seconds or until the PTA is completely solubilized to produce 3%PTA solution. Use a one milliliter syringe with a 22 gauge needle to draw up 0.5 milliliters of the 3%PTA solution, add four drops of the 3%PTA solution to the particles and vortex on the highest setting for 10 seconds.

Then, use a pair of self closing tweezers to pick up a TEM grid and dip it into the particle solution quickly three times. Let the grid dry for five hours, under a cover to reduce dust collection on the grid. Finally, perform TEM imaging of the sample, using high contrast and a 40 micron objective.

This figure represents the nanosponge synthesis method using a linear polyester copolymer containing pendant functional groups reacted with a diamine crosslinker to form discrete nanoparticles and solution. TEM imaging is used to characterize the precise dimensions of each set of nanosponges, this image contains nanosponges of 79 nanometers with the standard deviation of 12 nanometers. Nanosponge sizes were analyzed based on molecular weight of the polymer precursor and the amount of pendant functionality to evaluate the relationship between these two factors.

A trend of increasing nanosponge size is correlated with an increase in polymer molecular weight, independent of the pendant functionality. Nanosponge sizes were also analyzed based on the amount of pendant functionality and the amount of diamine crosslinker added to the reaction. Increasing both pendant functionality and crosslinker equivalent shows an increase in nanosponge sizes.

After watching this video, you should have a good understanding of how to utilize these synthetic parameters to design these nanoparticles to control the size and the morphology, exactly how you want to have them. We can tune the synthesis of these nanosponges with these important parameters:polymer molecular weight, apoxite concentration and the amount of crosslinker. After the development of this nanoparticle synthesis, this technique has been vital for our research lab to explore drug delivery of small hydrophobic compounds.

While attempting this procedure, it is very important to be accurate with measurements, as the stoichiometry and concentration are very important to the final product. Following this procedure, other methods for polymer network synthesis can be performed in order to answer questions related to the design of drug delivery platforms.