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

Summary

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.

Abstract

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.

Introduction

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 properties^1-3. 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 acid^5,6, 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 fashion^9,10. 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 system^10-12. Such platforms include: (1) MNPs/pNIPAM hybrid microgel particles ^13-15 and (2) macroscopic hydrogel scaffolds incorporating immobilized MNPs^16-18. 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 applications^21-23.

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.

Protocol

Note: The overall process of fabricating magnetic field-responsive gelatin microgels is illustrated in Figure 1A.

1. Preparing Solutions and Suspensions

1. 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.
2. Prepare a surfactant solution by dissolving 20 mg of poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (M_w = 2,900 Da; referred to as L64) in 200 ml of PBS to be at the concentration of 100 ppm.
3. Prepare a 15% (w/v) gelatin solution by dissolving 64.5 mg of gelatin in 0.43 ml of PBS. Vortex the solution and place it on water bath at 37 ^oC until it reaches a sol phase, where the solution becomes fluidic. Then, vortex the gelatin solution 2 - 3 times to ensure the homogeneity of the sample.
4. Preparation of p(NIPAM-co-AAm)/MNPs solution with a model drug (BSA):
   1. Disperse 10.75 mg of hydrophilic MNPs in 0.43 ml of PBS and then dissolve 12.9 mg of p(NIPAM-co-AAm) in the MNP suspension to make the concentration at 3% (w/v). The increased concentration of p(NIPAM-co-AAm) can be used to achieve an increased deswelling behavior of microgels.
   2. Use Texas-Red conjugated bovine serum albumin (TR-BSA; M_w ~66 kDa) as a model drug. Dissolve 0.5 mg of TR-BSA in the mixture of p(NIPAM-co-AAm)/MNPs.
5. Prepare mixtures of gelatin/p(NIPAM-co-AAm)/MNPs/BSA solution (0.86 ml) by adding mixture of p(NIPAM-co-AAm)/MNPs (0.43 ml) into the gelatin solution (0.43 ml) and then thoroughly vortex them to make a homogeneous mixture. Thus, the concentrations of polymers and MNP become half of the initial concentrations in the final mixture.

2. Emulsification

1. Pour 15 ml of silicone oil [polydimethylsiloxane (viscosity 350 cSt)] into a clean and sterile beaker.
2. Immediately add the pre-prepared aqueous mixtures of gelatin/p(NIPAM-co-AAm)/MNPs/BSA solution (0.86 ml) into the silicone oil and emulsify the aqueous mixture in the oil phase by stirring with a magnetic stirring bar at 900 rpm at 30 ^oC for 30 min.

3. Gelation and Transfer of Micro-droplets to an Aqueous Solution

1. Transfer the emulsion (~16 ml) from beaker into a 50 ml tube.
2. Cool down the tube for 10 min at 4 °C for gelation of the micro-droplets in the oil.
3. Fill the tube with the prepared L64 solution (at 4 ^oC) up to 50 ml and vigorously shake the tube. It may be possible that a portion of L64 surfactants would be within the microgels.
4. Centrifuge the tube for 20 min at 2,300 x g at 4 ^oC.
5. Regularly check for the presence of the pellet of gel particles on the side of the tube. If the particles are not seen, centrifuge for another 20 min at the same speed and temperature. Proceed to carefully remove supernatant without disturbing the pellet formed on the inner wall of the tube.
6. Repeat steps (3.3) through (3.5) once more. Each time, transfer the sample to a new tube to avoid the inclusion of any oil droplets in the microgel suspension. After this step, ensure that surfactants or oil droplets are not present in the sample suspension. However, the repeated separation steps may lead to loss of initial materials.

4. Covalent Crosslinking of the Microgels

1. Add 2 ml of genipin solution (prepared in section 1) to the pellet of gel particles and mix them well by vortexing the solution.
2. Quickly transfer the tube of the suspension in water bath at 23 ^oC to initiate a covalent crosslinking reaction during a desired crosslinking time (e.g., 5 - 120 min).
3. After crosslinking, immediately remove any excessive crosslinkers by discarding the genipin solution, resuspending the microgels in PBS, and centrifuging the tube for 20 min at 2,300 x g (4 ^oC). If needed, cautiously break apart formed pellet with a pipette tip. This washing step can be repeated up to 3 times if the genipin is still remaining in the solution.
4. Discard the supernatant and resuspend the microgels in PBS at a desired density (e.g., 5 × 10⁶ microgels/ml) by counting the number with a hemocytometer.
5. For microscopic observations, load the microgel suspension in the space between a slide glass and a cover slip and seal the boundary of the cover slip with epoxy resin.

5. Application of Alternating Magnetic Field for Triggering Drug Release

1. Place the tube with desired concentration of microgels in aqueous media into the chamber of magnetic coils. If necessary, insert a fiber optic temperature probe into the tube to monitor temperature change of the media during the application of AMF.
2. Apply high frequency (>100 kHz) AMF at a defined field strength (>5 kA/m) and for a specified duration. Following the application of AMF, centrifuge the sample tube for 20 min at 2,273 x g (4 ^oC) and collect the supernatant to quantify the amount of TR-BSA released from microgel to the surrounding media using spectrophotometry. The excitation and emission wavelengths for Texas Red are 584 nm and 612 nm, respectively.

Results

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 μm to 20 μm (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 ^oC f...

Discussion

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gelatin	Sigma-Aldrich, MO, USA	G2500	Gelatin type A, porcine skin
poly(N-isopropylacrylamide-co-acrylamide)	Sigma-Aldrich, MO, USA	738727	MW = 20,000, LCST = 34 - 38 °C
Silicone oil	Sigma-Aldrich, MO, USA	378372	Viscosity 350 cSt
Pluoronic L64	Sigma-Aldrich, MO, USA	435449	poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)
genipin	TimTec LLC, DE, USA	ST080860	MW = 226.23
Magnetic nanoparticles (MNPs)	Micromod Inc, Germany	79-00-102	nanomag-D-spio, 100 nm
TR-BSA	Life Technologies, NY USA	A23017	Albumin from Bovine Serum (BSA), Texas Red conjugate