자기 조립, 분해 Thermoresponsive Hydrogels 반응 압출, 마이크로, 통해 여러 길이 가늠 자에를 조작 하 고 전기

The overall goal of this protocol is to fabricate degradable versions of thermoresponsive hydrogels with controlled length scales and geometries. This method can help address key challenges in the scenario of biomedical materials such as facilitating long term, minimally invasive local drug delivery, creating responsive scaffolds for tissue engineering, or designing targeted nanotherapeutics. The main advantage of our technique is a rapid reverse biohydrazone chemistry which allows us to control the size and shape of the degradable hydrogels by changing the polymer precursor mixing geometry.

This flexibility lets us fabricate folic gels, microgels, nanogels, and nanofibers all with the same starting material. First, prepare about two milliliters each of hydrazide and aldehyde-functionalized thermoresponsive precursor polymer solutions with concentrations between five and 40%by weight and 10 millimolar phosphate-buffered saline. Use single barrel syringes to load the precursor polymer solutions into separate barrels of a 2.5 milliliter, 1:1 double barrel syringe.

Next, connect a 1.5 inch static mixer to a 2.5 milliliter 1:1 double barrel syringe. If desired, attach an 18 gauge or 20 gauge 1.5 inch needle to the static mixer. Then, cut from a soft, silicone rubber sheet of the desired thickness.

A piece about the size of a standard microscope slide. Use a punch set to punch at least one hole of the desired shape into the silicone rubber sheet. Place the silicone rubber mold on a clean glass slide, ensuring that all holes are backed by the glass.

Gently press the mold against the slide to fix it in place. Silicone rubber sheeting may also be used as backing to ensure good substrate adhesion. Co extrude the hydrazide and aldehyde functionalized polymer solutions through the static mixer into the mold wells until the wells are filled completely.

Cover the mold with a second clean, glass slide and wait for gelation to finish. Then, remove the top slide and use a spatula to separate the hydrogels from the silicone rubber mold. Lift the mold from the slide to recover the hydrogels.

Prior to the procedure, fabricate a microfluidic chip for the microparticle generation. Synthesize hydrazide and aldehyde functionalized poly NIPAM. To begin, prepare two milliliters each of 6%by weight solutions of hydrazide and aldehyde functionalized poly NIPAM in deionized water.

Load the polymer precursor solutions into separate five milliliter syringes. Next, prepare 150 milliliters of a 1%by weight solution of sorbitan monooleate in heavy paraffin oil. Load this solution in two standard 60 milliliter syringes.

Mount the four syringes in separate infusion syringe pumps. Next, connect a 45 centimeter length of silicon tubing to the microfluidic chip outlet. Place the end of the outlet tubing in a waste container.

Connect the polymer solution syringes to the polymer inlet channels on the microfluidic chip with 30 centimeters lengths of silicon tubing. Connect one paraffin oil solution syringe to the upstream oil inlet channel with silicon tubing. Connect the other oil syringe to both downstream oil inlet channels using silicon tubing and a U-shaped syringe joint.

Set the flow rates of the pumps equipped with oil syringes to between 1.1 and 5.5 milliliters per hour. Flow oil through the microfluidic chip for at least 30 minutes to prime the chip and to confirm that the chip is defect free. Then, simultaneously deliver both polymer solutions to the primed chip at 03 mL per hour.

Allow the flow of polymer and oil to equilibrate for 30 minutes to an hour. To prevent premature gelation of the precursor polymers at the microfluidic nozzle, it is essential to carefully prime the tubing and start the flow of polymer exactly at the same time to prevent back flow into either polymer reservoir. Once uniform gel microparticles are forming at the nozzle of the microfluidic chip, collect the particles in a plastic centrifuge tube with an active magnetic stir bar.

Stop the syringe pumps and the stir motor when the oil has been consumed. Once the gel microparticles have settle in the tube, decant the paraffin oil with a pipette. Add to the flask 10 milliliters of pentane for every 5 mL of microparticles.

Vigorously mix the emolution for about a minute. Allow the particles to settle for one to two hours before decanting the pentane with a pipette. Repeat this pentane wash at least five times.

Transfer the washed gel microparticles to a small, glass scintillation vial. Resuspend the particles in one to two milliliters of deionized water. To begin this procedure, prepare five milliliters each of 1%solutions of hydrazide and aldehyde-functionalized poly NIPAM in deionized water.

Heat five milliliters of the hydrazide-functionalized poly NIPAM solution to 70 degrees celsius while stirring. Monitor the solution as it becomes opaque and confirm that no visible precipitate forms. Then, add 0.25 milliliters of the aldehyde-functionalized poly NIPAM solution drop-wise to the heated hydrazide-functionalized poly NIPAM solution over the course of five to 10 seconds.

Stir the mixture for 15 minutes at 70 degrees celsius. Then, remove the mixture from the oil bath and allow it to cool to room temperature over night to obtain the nanogels for purification. Place the nano gel mixture in a 3500 kilodalton molecular weight cut off dialysis membrane.

Dialyze the nanogels against dionized water six times over six hour cycles to remove non-cross-linked polymers. If desired, lyophilize the purified nanogels for storage. To begin the gel nanofiber fabrication procedure, prepare one milliliter each of 15%by weight solutions of hydrazide and aldehyde-functionalized POEGMA in deionized water.

Then, prepare two milliliters of a 5%by weight solution of high moleculor weight polyethylene oxide in deionized water. Combine each POEGMA precursor solution with one milliliter of the polyethylene oxide solution. Load the precursor solutions into separate barrels of the double barrel syringe.

Next, connect a 1.5 inch static mixer and a blunt tipped 18 gauge needle to a 2.5 milliliter, 1:1 double barrel syringe. Mount the double barrel syringe on an infusion syringe pump. Then, mount an aluminum disc electrospinning collector 10 centimeters from the end of the needle.

Ground a high-voltage power supply at the collector and connect the power supply to the needle. Set the syringe pump to 0.48 milliliters per hour and the power supply to 10 kilovolts. Simultaneously start the syringe pump and the power supply to initiate electrospinning.

Continue until the desired thickness is reached or the precursors are exhausted. After nanofiber production has finished, turn off and disconnect the electrospinning assembly. Soak the collected gel nanofibers in deionized water for 24 hours to remove the polyethylene oxide.

Double barrel syringe delivery produced hydrazone cross-linked POEGMA bulk hydra-gels with a range of mechanics, gelation times, and transparencies depending on the precursor polymer concentration, the reactive functional group density, and the number of ethylene oxide repeat units on the monomers. The flow rates of both the paraffin oil and the polymer precursor solutions affected the sizes of hydrozone cross-linked poly NIPAM gel microparticles fabricated with the microfluidic device. Reversible temperature dependent swelling was observed with hot stage optical microscopy.

The gel micro particles degraded to their oligomeric precursors over time. The sizes of self-assembled hydrazone cross-linked poly NIPAM nanogels varied with the aldehyde to hydrazide functionalized polymer precursor-mass ratio but remained highly mono dispersed. As the mass ratio increased, less thermal deswelling was observed.

Like the gel microparticles, the nanogels degraded to the oligomeric precursors over time. Hydrazone crosslinked POEGMA hydrogel nanofibers with diameters on the order of 300 nanometers were produced by reactive electrospinning. Nanofiber hydration was about two orders of magnitude faster than hydration of a bulk gel with the same polymer composition.

The nanofibers retained their nanofibrous morphology for over eight weeks before degrading under physiological conditions. Faster degradation occurred in acidic environments. The nanofibers were mechanically robust in both the dry and swollen states.

After watching this video, we hope you have a good understanding of how to create degradable, thermo-responsive hydrogels by controlling the geometry at which the gel precursor polymers are made. Remember to ensure the gelation time of the polymer precursor matches the needs of each particle. Gelation that is too fast will inhibit the microgel and the nanofiber fabrication.

Gelation that is too slow will result in the fully congeeled morphologies. These techniques pave the way for researchers in the areas of drug delivery and tissue engineering. and design well-defined hydrogel scaffolds for controlling the rate or location of drug release or stimulating tissue regeneration.