This overall goal of this protocol is to present a facile method for fabrication of magnetothermally responsive nanoparticle microgel hybrids, and to demonstrate a proof of concept on the use of the microgel hybrids for controllable drug release. The main advantages of this protocol are that the process is relatively simple and it confers the microgel to exhibit the magnetothermally responsive characteristic by simply entrapping magnetic nanoparticles and thermoresponsive PNIPAM copolymers within the gelatin matrix. This allows for drug released in response to an alternating magnetic field, which is induced by the swelling of the PNIPAM copolymer associated with the increasing temperature of the nanoparticles as the nanoparticles respond to the magnetic waves.
The implications of this technique extend toward developing on-demand drug delivery system because this controllable drug release platform is responsive to multiple on and off cycles induced by non-invasive external stimuli. The process of fabricating magnetic field responsive gelatin microgels begins with preparing the solutions in suspension. Basic solution preparation is described in the text protocol.
When preparing the gelatin solution, keep it in a water bath until it reaches the fluidic phase. Then homogenize it with a vortex. With all the solutions prepared mix equal volumes of gelatin solution and the PNIPAM copolymer containing magnetic nonaparticles and vortex the mixture thoroughly.
The next step is to emulsify the aqueous mixture in silicon oil and then apply a surfactant coat. Pour 15ml of silicone oil into a sterile beaker and immediately add 0.86ml of the prepared mixture. Then stir the mixture at 900 rpm at 30 degrees Celsius for half an hour.
After producing the emulsion, transfer it to a 50ml tube and cool it down at four degrees Celsius for 10 minutes. Microdroplets will now form in the oil. Next, mostly fill the tube with Pluronic L-64 surfactant solution, Cool to four degrees Celsius, and shake the tube vigorously so the L-64 molecules get adsorbed into gel surface and stabilize the microgel suspension.
Then centrifuge the tube for 20 mintues at 2300 Gs and at four degrees Celsius. After 20 minutes, examine the tube for a pellet of gel particles. Repeat the centrifugation cycle until one can be seen.
Then carefully remove the supernatant and fill the tube to 45 ml with cold L-64 solution. Shake the tube hard to mix the pelleted microgels into the surfactant. And transfer the suspension to a new 50ml tube.
Then repeat the centrifugation process and collect the pellet of microgels again. Now, to ensure that no surfactants or oil droplets remain in the sample suspension thoroughly aspirate the supernatant. The next step to preparing the microgels is to crosslink their gelatin matrix.
To begin, suspend the pellet in 2ml of the prepared Genipin solution. Use a vortex. Then quickly transfer the suspension to a 23 degrees Celsius water bath to initiate the covalent crosslinking reaction.
The reaction can be run for five minutes to two hours. Once completed, removed the excess crosslinkers. Centrifuge the sample for 20 minutes at 2300 Gs and at four degrees Celsius as before.
Then, aspirate and discard the supernatant. Repeat the washing step in PBS a total of three times, breaking up the pellet mechanically with a pipette tip if needed. Complete removal of the unreacted excess crosslinkers is critical.
After the last wash, bring the microgels up in PBS to the desired density. Measure their density using a hemocytometer. The fabricated microgels are stable for up to four weeks when stored at four degrees Celsius, provided that there is no degrading agent, such as collagenase.
To characterize the microgels by microscopy load them on a slide and seal them under a cover slip with epoxy resin. The final step of the procedure is to measure drug release from the microgels. Place the tube with the desired concentration of microgels into the chamber of magnetic coils.
Then, insert a fiber optic temperature probe to monitor temperature change during the application of the alternating magnetic field. Now, apply high frequency alternating magnetic field at a defined field strength and for a specified duration. Following the treatment, centrifuge the tube for 20 minutes at 2300 Gs and at four degrees Celsius.
Collect the supernatant to quantify the amount of drug released from the microgels. In this case, use spectrophotometry to detect the texas red. The fabricated microgels should exhibit a well-characterized spherical morphology and colloidal dispersability with diameters between five to 20 microns.
Either fluorescent superparamagnetic nanoparticles or fluorescently labelled BSA can be used to confirm proper encapsulation within the microgel. The entrapment of PNIPAM copolymer in the gelatin microgel matrix enables it to exhibit a temperature-dependent volume change. Increasing the temperature of the media from 22 to 42 degrees Celsius resulted in deswelling of gelatin microgels by about 40%In contrast, only 10%deswelling was observed in gelatin microgel without PNIPAM copolymer.
When superparamagnetic nanoparticles were incorporated into the gelatin PNIPAM copolymer microgel, the microgel increased in temperature when exposed to the appropriate alternating magnetic field. Measured TRBSA release from these microgels was about 35%Without incorporating the copolymer into the microgels, the release of TRBSA was significantly lower. Only around 10%Thus, the release of BSA in response to the alternating magnetic field was likely due to deswelling associated with the shrinkage of PNIPAM copolymer chains within the microgel.
After watching this video, you should have a good understanding of how to fabricate biodegradeable magnetic field responsive nanoparticle microgel hybrids that enable controllable drug release using a simple water-in-oil emulsification method. Once mastered, this technique can be done in three hours if it is performed properly. This protocol also can be modified to fabricate microgel hybrids that are responsive to other stimuli, such as near infrared light.
By simply incorporating near infrared responsive gold nanoparticle with gelatin matrix.
