Name: alternating magnetic field-responsive hybrid gelatin microgels for controlled drug release
Uploaded: 2016-02-13T15:00:00
Description: Watch this Scientific Journal Video about Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release at JoVE.com

This study was supported by Farris Family Innovation Award and NIH 1R01NR015674-01 to MK. The authors thank Josep Nayfach (Qteris, Inc) for providing an electro-magnetic generator system as well as his technical consultation. The authors also thank Huan Yan (LCI &#38; Chemical Physics Interdisciplinary Program, Kent State University) for her technical assistants.

Note: The overall process of fabricating magnetic field-responsive gelatin microgels is illustrated in Figure 1A.
1. Preparing Solutions and Suspensions
<ol>
	<li>Prepare a crosslinker genipin (1% w/v) solution by dissolving 20 mg of genipin in 2 ml of phosphate buffered saline (1x PBS; pH 7.4). Vortex the solution and place in a 50 oC water bath for 2 hr to completely dissolve the solution.</li>
	<li>Prepare a surfactant solution by dissolving 20 mg of poly(ethyl...

<ol>
	<li>Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+in+drug+delivery+and+tissue+engineering:+one+laboratory's+experience.">Biomaterials in drug delivery and tissue engineering: one laboratory's experience.</a> Acc. Chem. Res. 33, 94-101 (2000).</li><li>Rivest, C. 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The technology described here demonstrates a proof of concept on the use of nanoparticle-microgel hybrids for magneto-thermally triggered drug release. This was achieved by physically entrapping MNPs and p(NIPAM-co-AAm) chains within a microscale three-dimensional gelatin network crosslinked by genipin. The magnetic field-responsive platform was sufficient to generate heat within the microgel in response to a remotely applied AMF, which in turn triggered the release of a model drug, BSA.
<p class="jove_conte...

Stimuli-responsive drug delivery systems that enable a tightly controlled drug delivery in response to either endogenous or exogenous stimuli (e.g., temperature or pH) have been extensively investigated as new types of smart soft devices for drug delivery. Microscale hydrogels have been widely employed as a drug delivery platform in that they confer controllable and sustainable drug release profiles as well as tunable chemical and mechanical properties1-3. In particular, the colloidal microgels exhibit many advantages as a vehicle for drug delivery due to their rapid responsiveness to external stimuli and suitable injectability to local tissue in a minimally invasive manner4. The poly(N-isopropylacrylamide) (pNIPAM) or its copolymers have been widely adopted in synthesizing thermo-responsive microgels by grafting pNIPAM with biodegradable/ biocompatible polymers including gelatin, chitosan, alginate acid, or hyaluronic acid5,6, in which a phase transition characteristic of pNIPAM at its lower critical solution temperature (LCST) can be used as a trigger of drug release7. We recently demonstrated a fabrication of biodegradable, gelatin-based thermo-responsive microgel by incorporating poly(N-isopropylacrylamide-co-acrylamide) [p(NIPAM-co-AAm)] chains as a minor component within three-dimensional gelatin networks8. The gelatin/p(NIPAM-co-AAm) microgel exhibited a tunable deswelling to temperature increase, which positively correlated to the release of bovine serum albumin (BSA).
During the last several years, there have been increasing efforts to develop a magnetically responsive drug delivery platform that can trigger the release of drug in an on-demand fashion9,10. The basic principle for the synthesis of magnetically responsive drug delivery platform utilizes the characteristic of superparamagnetic nanoparticles (MNPs) to generate heat when they receive a high frequency alternating magnetic field (AMF), which triggers a temperature-sensitive drug release. This holds promise for future clinical applications in that this system can target deep into the tissue, enables a non-invasive and remotely controlled drug release and can be combined with hyperthermia treatment and magnetic resonance imaging system10-12. Such platforms include: (1) MNPs/pNIPAM hybrid microgel particles 13-15 and (2) macroscopic hydrogel scaffolds incorporating immobilized MNPs16-18. The pNIPAM-based microgel platforms demonstrated a finely-tunable volume phase transition responsiveness to magneto-thermal stimuli. However, they still rely on complex and sophisticated techniques in the fabrication and the use of pNIPAM polymers with a high content can be potentially cytotoxic to cells19, which may limit their in vivo applications. The macroscopic scaffolds exhibit a relatively slow response to external stimuli and require an invasive surgical transplantation compared to colloidal microgels.
The water-in-oil emulsification has been the standard method to produce submillimeter or micrometer-sized gel particles20. At the water-oil interface of the emulsion, microgel particle forms a spherical shape due to the minimization of surface energy of the water droplet under mechanical shear force. This method allows the production of a large amount of aqueous spherical gel droplets in a simple fabrication procedure and has been successfully adopted for fabricating gelatin-based microgels for drug delivery applications21-23.
Here, we present a facile method to synthesize a magnetothermally responsive gelatin-based microgels for drug delivery application by employing the water-in-oil emulsification method. This was achieved by physically incorporating iron oxide MNPs and p(NIPAM-co-AAm) chains as a minor component within a spherical microscale gelatin network that is covalently crosslinked by a naturally-derived crosslinker genipin, in conjunction with a high frequency alternating magnetic field (AMF) application system.

<table border="0" cellpadding="0" cellspacing="0" width="706">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelatin</td>
			<td style="width:204px;">Sigma-Aldrich, MO, USA</td>
			<td style="width:119px;">G2500</td>
			<td style="width:167px;">Gelatin type A, porcine skin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">poly(N-isopropylacrylamide-co-acrylamide)&#160;</td>
			<td style="width:204px;">Sigma-Aldrich, MO, USA</td>
			<td>738727</td>
			<td>MW=20,000, LCST=34-38 oC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silicon oil</td>
			<td style="width:204px;">Sigma-Aldrich, MO, USA</td>
			<td>378372</td>
			<td>Viscosity 350 cSt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pluoronic L64</td>
			<td style="width:204px;">Sigma-Aldrich, MO, USA</td>
			<td style="width:119px;">435449</td>
			<td>100 ppm poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">genipin</td>
			<td style="width:204px;">TimTec LLC, DE, USA</td>
			<td>ST080860</td>
			<td>Mw = 226.23;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnetic nanoparticles (MNPs)</td>
			<td style="width:204px;">Micromod Inc, Germany</td>
			<td>79-00-102</td>
			<td>nanomag-D-spio, 100 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TR-BSA</td>
			<td style="width:204px;">Life Technologies, NY USA</td>
			<td>A23017</td>
			<td>Albumin from Bovine Serum (BSA), Texas Red conjugate</td>
		</tr>
	</tbody>
</table>

alternating magnetic field-responsive hybrid gelatin microgels for controlled drug release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

When the protocol is performed correctly, the fabricated microgels should exhibit a well-characterized spherical morphology and colloidal dispersibility with diameters in the range between 5 &#956;m to 20 &#956;m (Figure 1B and C). Either fluorescent MNPs or fluorescent BSA can be used to confirm whether MNPs or drugs (BSA in this study) are properly encapsulated within the microgel (Figure 1D). The fabricated microgels can be stable and stored at 4 ...

Watch this Scientific Journal Video about Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release at JoVE.com

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

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.

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Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...

Low-Cost, Volume-Controlled Dipstick Urinalysis for Home-Testing

Dipstick urinalysis provides quick and affordable estimations of multiple physiological conditions but requires good technique and training to use ...

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...