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Injectable Macroporous Ferrogel Microbeads with a High Structural Stability for Magnetically Actuated Drug Delivery Bom Yi Shin, Bong Geun Cha, Ji Hoon Jeong, and Jaeyun Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06444 • Publication Date (Web): 01 Sep 2017 Downloaded from http://pubs.acs.org on September 2, 2017

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ACS Applied Materials & Interfaces

Injectable Macroporous Ferrogel Microbeads with a High Structural Stability for Magnetically Actuated Drug Delivery Bom Yi Shin,1, ‡ Bong Geun Cha,1, ‡ Ji Hoon Jeong,2 Jaeyun Kim1,3,4∗ 1

School of Chemical Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea

2

School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea

3

Department of Health Sciences and Technology, Samsung Advanced Institute for Health

Science & Technology (SAIHST), Sungkyunkwan University, Suwon 16419, Republic of Korea 4

Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University (SKKU),

Suwon 16419, Republic of Korea ‡

∗

These authors contributed equally.

Corresponding author: Jaeyun Kim, [email protected]

1 Environment ACS Paragon Plus


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ABSTRACT: Macroporous hydrogels are an attractive material platform that can provide shortened interfacial diffusion pathways and high biomacromolecule loading. Recently, macroporous ferrogels have shown high potential for use in the on-demand delivery of bioactive molecules, resulting from their reversible and large volumetric deformation upon magnetic stimulation. However, these macroporous ferrogels require surgical placement in the body due to their large size; an injectable form of macroporous ferrogels has not yet been reported. In this study, injectable macroporous ferrogel microbeads loaded with iron oxide nanoparticles have been prepared based on alginate microbeads for on-demand drug release. A simple solvent exchange and subsequent covalent crosslinking of the alginate chains in hydrogel microbeads induced a high polymer density on the hydrogel network and led to enhanced mechanical properties even after the generation of macropores in the microbeads. The macroporous ferrogel microbeads exhibited good mechanical stability and were stable during needle injection. The increased loading of large biomolecules due to the macroporosity of the microbeads and their large reversible volumetric deformation response to the external magnetic field enabled their potential for use in the on-demand delivery of drugs of assorted sizes by magnetic actuation. As a result of their structural stability, injectable size, and ability for on-demand drug delivery, ferrogel microbeads have promising potential for application in many biomedical fields.

KEYWORDS: macroporous hydrogel, ferrogel, microbead, on-demand delivery, magnetic actuation


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INTRODUCTION Hydrogels are soft functional materials containing large amounts of water, which have a wide range of bioapplications due to their hydrophilic and biocompatible properties.1-6 Macroporous hydrogels with well-defined macroporous structures can provide shortened interfacial diffusion pathways that enhance the adsorption and release of guest molecules including small molecules, DNA, proteins, and even cells, compared with nanoporous hydrogels.7-10 Macroporous structures in hydrogels can be created by several approaches, such as the template-mediated method that generates macropores by removing sacrificial templates, the cryogelation method that creates pores by the formation and sublimation of ice crystals upon freezing,11 and the reaction-induced phase separation technique that involves the polymerization of monomers and crosslinkers using an inert diluent that is removed after the reaction to generate macropores.12,13 Using these methods, macroporous hydrogels consisting of diverse materials, such as alginate, poly(lactic-coglycolic acid) (PLGA), and gelatin, have been successfully prepared and utilized in various applications including drug delivery, gene delivery, angiogenesis, and immunotherapy.14,15 However, these macroporous hydrogels are fabricated in a pre-shaped bulk form; hence, they can only be introduced into the body via a surgical procedure. Therefore, the development of small macroporous hydrogels (e.g., macroporous hydrogel microbeads) that can be introduced into the body in a minimally invasive manner, such as through needle injection, is required. Despite the high demand for this type of hydrogel, the preparation of injectable macroporous hydrogel microbeads has not yet been reported. It is usually difficult to form large amounts of macropores within small hydrogel microbeads while maintaining their structural stability, because of their large water content, which prevents the formation of stable macroporous hydrogel microbeads after application of the aforementioned macropore generation methods. Even if it were possible



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to prepare macroporous hydrogel microbeads, the weak macroporous structures could be easily destroyed during handling of the microbeads, such as in washing steps. Therefore, the preparation of macroporous hydrogel microbeads with a high pore connectivity and a high mechanical stability that can be maintained during the injection process remains a challenge. Hydrogel-based, on-demand drug delivery systems have become a major research area for the application of hydrogels. Among several material platforms, ferrogels have a high potential for use in on-demand drug delivery as well as in actuation and sensing. Ferrogels are composed mainly of a polymer matrix embedded with magnetic micro- and nano-particles.16,17 Upon the application of an external magnetic field, the polymer matrix of the ferrogel can deform due to the magnetic force generated by the embedded magnetic particles, which would allow actuation and magnetically driven drug release on demand. Macroporous ferrogels have recently been shown to exhibit larger volumetric deformations than conventional nanoporous ferrogels due to the existence of macropores; these deformations could induce significant local convections in the gel, resulting in the rapid release of drugs over a short time period upon the application of a magnetic field.18 More recently, to overcome the insigniﬁcant deformation displayed by macroporous ferrogels that are optimally sized for implantation (e.g., a 2 mm thickness), a new biphasic ferrogel with a magnetic nanoparticle gradient capable of large deformations even at a small gel size has been presented.19,20 However, these macroporous ferrogels require surgical placement in the body and there have not yet been any reports of macroporous ferrogels with an injectable microbead morphology, which are strongly preferred by patients since they would not require surgical implantation. Here, we demonstrate for the first time, the preparation of alginate-based macroporous ferrogel microbeads that can be injected via a needle. Macroporous alginate ferrogel microbeads


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with a well-defined interconnected porous structure and good mechanical stability were prepared via a template method (Scheme 1a). Ca2+-crosslinked alginate microbeads embedded with porogen microparticles and magnetic nanoparticles were size-reduced by a simple solvent exchange,21 and the closely rearranged alginate networks were then covalently crosslinked to prevent collapse of the microbead structure during the removal of the porogen to form macropores. Due to their macroporosity and high structural stability, the macroporous ferrogel microbeads showed a high deformation in response to an external magnetic field without a structural collapse (Scheme 1b), enabling their use in the on-demand delivery of guest macromolecules.

RESULTS AND DISCUSSION To prepare macroporous alginate hydrogel microbeads based on a template method using CaCO3 microparticles as sacrificial templates, it was hypothesized that stable crosslinking of the alginate chains in the hydrogel microbeads would lead to mechanically stable macroporous hydrogel microbeads even after the removal of CaCO3 microparticles. However, as the alginate concentration in the Ca2+-crosslinked hydrogel is usually around 2 wt% due to its high viscosity in the aqueous solution; the alginate crosslinking density might not be high enough to maintain the macroporous structure after removal of the sacrificial template. In addition, the use of a calcium chelator, ethylenediaminetetraacetic acid (EDTA), to generate macropores in microbeads by removing the incorporated CaCO3 sacrificial template, would also dissociate calcium-crosslinked alginate microbeads. To circumvent these two limitations in our strategy, we applied a remodeling and subsequent crosslinking (RsC) process by conducting a gradual solvent exchange for the Ca2+-crosslinked alginate microbeads followed by covalent-crosslinking of the



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alginate chains.21 Through the gradual exchange of the original aqueous media with the less polar, hydrophilic solvent, isopropanol, size-reduced alginate microbeads were formed from the highly dense alginate network, due to the decrease in alginate solubility in the exchanged solvent (Figure 1a, b). The dense alginate microbeads that were further ionically-crosslinked with Ba2+ ions showed significantly improved mechanical properties compared to the original alginate microbeads. Subsequently, we bridged the alginate chains with covalent bonding, using hexamethylenediamine to crosslink the activated hydroxyl group of the alginate matrix, allowing it to resist EDTA treatment and to maintain the macroporous hydrogel structure after the removal of the CaCO3 template microparticles. The alginate chains drew closer to each other in the dense alginate microbeads after the RsC process, and these more closely packed alginate chains facilitated the subsequent covalent crosslinking between the alginate chains in the dense alginate microbeads. After the covalent crosslinking, the calcium carbonate-encapsulated alginate hydrogel microbeads were treated with a 50 mM EDTA solution to remove the CaCO3 microparticles. The optical microscope images of the alginate microbeads during EDTA treatment clearly show that the CaCO3 microparticles were gradually removed from the outside to the inside over time (Figure 1c–f). In contrast, the alginate microbeads without covalent bonding collapsed to dissolve in solution within 10 min during the EDTA treatment, due to breakage of the ionic bridge of the alginate chains resulting from the dissociation of Ca2+ and Ba2+ions from the alginate chains. After the complete removal of the templates, the macroporous alginate microbead surface was rough (Figure 1g, h) in comparison to the smooth surfaces of the original alginate microbeads (Figure 1i), showing the generation of macropores on the surface and over the matrix after the removal of the template microparticles. Higher macroporosity and increased roughness on the surface of the alginate microbeads were achieved by the use of a


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larger amount of the CaCO3 templates, indicating that the macropore volume could be controlled simply by changing the amount of template (Figure S1). The covalent crosslinking of alginate chains in the ionically-crosslinked alginate microbeads is an important step in the production of macroporous hydrogel microbeads. The detailed procedures and the molecular structure of the alginate chains during covalent crosslinking and EDTA treatment are shown in Figure 2a and b. For the covalent crosslinking of the alginate chains in the hydrogel microbeads, the hydroxyl groups in the alginate were first activated with cyanogen bromide in basic conditions, at a pH above 10, and the activated cyanate ester groups in the alginate chains were subsequently crosslinked using hexamethylenediamine as a crosslinker (Figure 2b). Figure 2c–e shows the morphological change in the CaCO3incorporated alginate microbeads prepared without covalent crosslinking and after covalent crosslinking was performed for different pH values. When there was no additional covalent crosslinking in the alginate microbeads, the EDTA treatment resulted in the complete dissolution of the CaCO3 microparticles, as well as the Ca-crosslinked alginate microbeads, indicating that covalent crosslinking is required to maintain the stability of the alginate microbeads during the removal of the CaCO3 templates (Figure 2c). When covalent crosslinking was conducted at a lower pH, which did not provide sufficient activation and covalent crosslinking of the alginate chains, a portion of the alginate microbeads showed significantly swelling even after 5 min of EDTA treatment (Figure 2d). After 1 h of EDTA treatment, an enlarged alginate microbead population was seen to co-exist with the smaller alginate microbeads. Because the hydroxyl groups in the alginate were not fully activated at a lower pH, these alginate microbeads could not maintain the reduced size that was achieved from the RsC process during treatment with the EDTA solution. At a higher pH, the microbead size was maintained even after the removal of the



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CaCO3 microparticles with 1 h of EDTA treatment. The swelling ratios of the resulting microbeads prepared in lower and higher pH were 37 and 26 %, respectively, which is consistent with the microscopic observation as shown in Figure 2d and e. These results indicate that the covalent crosslinking was sufficient to maintain the morphology of the alginate microbeads and thus to prepare uniform, monodisperse macroporous alginate microbeads via the sacrificial template removal method (Figure 2e). The RsC process to reduce the size of the hydrogel microbeads and to shorten the distance between the alginate chains in the hydrogels is also crucial for the generation of a stable macroporous structure in the hydrogel microbeads after the removal of the CaCO3 microparticle templates. To investigate their structural stability, the macroporous alginate microbeads were freeze-dried and observed using scanning electron microscopy (SEM) (Figure 3). The size of the CaCO3 microparticle templates was around 20 µm (Figure 3a). The macroporous alginate microbeads prepared without the RsC process show a collapsed and contracted structure upon lyophilization due to the low mechanical stability of the hydrogel matrix in the presence of macropores generated by the removal of the CaCO3 microparticles (Figure 3b). In contrast, the microbeads obtained after the RsC process and subsequent EDTA treatment clearly show the successful generation of many macropores on their surface after the removal of the CaCO3 microparticles, and their macroporous structures were maintained even after lyophilization (Figure 3c, d). This is attributed to the increased density of the alginate matrix between macropores. In our previous work, it was found that the density of alginate in microbeads increases around 25-fold after the RsC process.21 These findings indicate that both size-reduction of alginate microbeads via the RsC process and subsequent covalent crosslinking of the alginate chains are required to maintain the stable macroporous structure of the alginate microbeads.


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For application of the macroporous alginate microbeads in an on-demand delivery system, we hypothesized that the encapsulation of magnetic nanoparticles in the hydrogel matrix could lead to a high volumetric deformation of the macroporous microbeads upon application of an external magnetic field. To test this hypothesis, superparamagnetic iron oxide nanoparticles were incorporated into alginate microbeads during the fabrication step, and macroporous magnetic alginate microbeads were prepared and designated as macroporous ferrogel microbeads. Energy dispersive X-ray spectrometry (EDS) analaysis of the ferrogels microbeads shows that there were no remaining Ca and Ba ions in the resulting ferrogels microbeads while a high amount of Fe was included (Figure S2). The quantitative analysis of Fe by inductively coupled plasma mass spectrometer (ICP-MS) shows that high amounts of iron oxide nanoparticles were embedded in the ferrogels microbeads (0.026 µg Fe/microbead). To investigate the magnetic actuation of the prepared macroporous ferrogel microbeads, an external magnetic field was applied to nonmacroporous and macroporous ferrogel microbeads and their volume changes were analyzed (Figure 4). Non-macroporous ferrogel microbeads showed almost no collective volume change upon exposure to the external magnetic field (Figure 4a). In contrast, the macroporous ferrogel microbeads exhibited a significant collective volume change upon exposure to the external magnetic field and clear deformation of individual microparticles was observed from microscopic images (Figure 4b). The large volume change of the macroporous ferrogel microbeads is attributed to the transient deformation of macropores upon the application of a magnetic field. The original volume was recovered after the removal of the magnetic field and multiple magnetic actuations, indicating that the covalent bonding-assisted macroporous alginate structure was stable enough to resist stress during magnetic actuation.



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We next investigated the loading efficiency of a model protein within alginate microbeads with and without macropores to determine their potential use as delivery vehicles. Ovalbumin (OVA) was physically adsorbed onto non-macroporous and macroporous microbeads at a pH of 4.5. The molecular weight of ovalbumin is 45 kDa and its estimated 3-dimensional size is 7.0 × 3.6 × 3.0 nm.22 The adsorption kinetics showed that the OVA was adsorbed within one hour, probably via electrostatic interactions between the slightly positively charged OVA at a pH of 4.5 and the strongly negatively charged alginate (Figure S3a). The amount of OVA adsorbed onto the macroporous microbeads was four times higher than onto the nonmacroporous microbeads (Figure S3b), indicating that the macropores significantly increased the loading efficiency of the biomacromolecules resulting from their high surface area and enhanced diffusion pathway. We further tested the loading of 13-nm gold nanoparticles that are much larger in size than OVA proteins. By increasing the number of macropores in the alginate microbeads, using increased amounts of the CaCO3 template, the adsorption of gold nanoparticles was increased (Figure S4), indicating that the macroporous microbeads have the potential to be used for post-loading of large biomolecules due to their macropores and the negatively charged alginate matrix. Based on the stable magnetic actuation displayed by the macroporous ferrogel microbeads and the high loading efficiency of biomacromolecules within these microbeads, we tested to see if the release of guest molecules loaded in macroporous ferrogel microbeads could be controlled by magnetic actuation. As a model molecule, fluorescein isothiocyanate (FITC)labeled dextran with 70 kDa molecular weight and 10 nm diameter,23 was loaded on the macroporous ferrogel microbeads by physical adsorption, and its release from the microbeads upon the repeated application of magnetic fields was measured (Figure 5a). A solid neodymium


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magnet was used for the application of magnetic field to macroporous ferrogel microbeads both in vitro and in vivo experiments. The strength of magnetic field measured on the surface of the solid magnet was 500 mT. The simulation data shows that fairly uniform magnetic field could be generated inside of well plate upon the application of solid magnet on the bottom of the plate (Figure S5). The magnetic field was applied for one minute with repeated actuations every 10 min. The typical slow release of dextran from the macroporous ferrogel microbeads was observed without the application of a magnetic field. In contrast, magnetic actuation resulted in a burst release of the dextran molecules. Furthermore, this burst release occurred only upon the application of an external magnetic field and the release rate returned to the normal slow release profile similar to that of the control after removal of the magnetic field. The on-demand release of the larger protein bovine serum albumin (BSA) from macroporous ferrogel microbeads by magnetic stimulation was also observed (Figure 5b). This on-demand release of model molecules of diverse sizes is attributed to the convection generated by high volumetric deformations in macropores upon the application of an external magnetic field.18 To further test if this on-demand release system could be applied as a drug delivery system, the on-demand release of an anti-cancer drug to cancer cells was investigated (Figure 5c). Mitoxantrone, a positively charged anti-cancer drug, has a high affinity for the negatively charged alginate chains via electrostatic interactions, which results in a high loading efficiency (2.45 mg mitoxantrone/g dried ferrogels microbeads) and a slower release of the drug from the alginate hydrogel despite its small molecular weight.24 Compared to untreated control cells without any treatment, the addition of macroporous ferrogel microbeads loaded with mitoxantrone showed a 50% reduction in cell viability. In contrast, magnetic actuation resulted in a further decrease, reducing cell viability to 30%, indicating that the active release of bioactive



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molecules could be easily achieved with the macroporous ferrogel microbeads. Furthermore, in contrast to macroscale ferrogels that require surgical placement, the macroporous ferrogel microbeads can be easily injected subcutaneously into a model mouse through needle injection (Figure 5d). Although we have demonstrated that the resulting ferrogels microbeads can be injected via a needle in the animal study, smaller sized microbeads are more desirable in the potential application to human patients. Among various methods to prepare alginate microbeads, recent microfluidic approaches show most promising results to fabricate uniform alginate microbeads smaller than 100 µm in a continuous way,25-27 which may offer an improved route to fabricate much smaller macroporous ferrogels microbeads suitable for the patient-friendly injection. Along with their structural stability and ability for on-demand drug delivery, the injectable ferrogel microbeads will have a high potential for application in many biomedical fields.

CONCLUSION In conclusion, the fabrication of macroporous ferrogel microbeads via a templated method using sacrificial CaCO3 microparticle templates has been demonstrated. A simple solvent exchange and subsequent covalent crosslinking of the polymer chains in hydrogel microbeads induced an ultrahigh density and enhanced mechanical properties even after the generation of large macropores, which could not be achieved in a conventional alginate hydrogel. The macroporous ferrogel microbeads displayed increased loading of large biomolecules, due to their macroporosity, and a large, reversible volumetric deformation response to an external magnetic field, enabling their potential for use in the on-demand delivery of a variously sized drugs by magnetic actuation.


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MATERIALS AND METHODS Materials. The following materials were purchased and used without further purification. Alginic acid sodium salt from brown algae, calcium chloride dehydrate, cyanogen bromide, hexamethylenediamine, sodium hydroxide, ammonium hydroxide solution, iron(III) chloride hexahydrate, iron(II) chloride tetrahydrate, sodium citrate tribasic dehydrate, albumin from chicken egg white and fluorescein isothiocyanate-dextran were purchased from Sigma-Aldrich (St. Louis, MO, USA). Barium chloride dehydrate was purchased from Kanto Chemical (Tokyo, Japan). Isopropyl alcohol and ethylenediaminetetraacetic acid (EDTA) were purchased from Samchun Chemical (Seoul, Korea). Alexa Fluor® 680 dye was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Preparation of CaCO3 microparticles. Equal volumes of 0.5 M solutions of calcium chloride and sodium carbonate were rapidly mixed and vigorously stirred for 30 min at room temperature. The reaction mixture was then centrifuged at 4,000 rpm for 5 min. The precipitated CaCO3 microparticles were thoroughly washed with water and ethanol three times and then air dried. Preparation of iron oxide nanoparticles. Iron oxide nanoparticles were prepared by a coprecipitation method. In a typical reaction, 0.86 g FeCl2 and 2.35 g FeCl3 (a 1:2 ratio of Fe2+/Fe3+) were mixed in 40 mL of water and vigorously stirred. Then, 5 mL of NH4OH was slowly dropped into the reaction mixture and stirred for a further 30 min. After the reaction, 5 mL of a 0.5 g/mL sodium citrate solution was added and the mixture was stirred for 90 min. The iron oxide nanoparticles were then centrifuged, washed with DI water, and redispersed in water. Preparation of alginate microbeads loaded with CaCO3 microparticles and iron oxide nanoparticles. An alginate mixture solution was prepared by combining alginate solutions (3 wt% in deionized water) with designated amounts of 0.15 g/mL CaCO3 microparticles and 0.5



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mg/mL iron oxide nanoparticles. Alginate microbeads were formed in a spherical shape by extrusion of the mixture solution through a 24-gauge blunt needle using a syringe pump (Pump 11 Series, Harvard Apparatus) into a 100-mM calcium chloride solution. The needle was connected to a high voltage power supply (230-30R, Bertan) that supplied 12 kV, and the calcium solution bath was grounded. The microbeads were immediately formed by Ca2+crosslinking of the alginate chains. The microbeads were washed with 0.9 w/v% sodium chloride (saline) solution several times and dispersed in phosphate buffered saline (PBS). Preparation of macroporous ferrogel microbeads. The dispersion media (PBS) of the alginate microbeads loaded with CaCO3 microparticles and iron oxide nanoparticles was gradually exchanged to isopropyl alcohol (IPA) while under vortex. The size-reduced alginate microbeads were immediately crosslinked with a 100-mM barium chloride solution for 30 min, resulting in fixing of the size-reduced, condensed form of the microbeads. To induce covalent crosslinking of the alginate chains, a cyanogen bromide solution (0.236 M) was initially added to the alginate microbeads dispersed in a 150-mM calcium chloride solution to give 5 equivalents of cyanogen bromide per monosaccharide unit of alginate. Next, the pH of the solution was quickly adjusted to 10 by the addition of 5 M sodium hydroxide. The microbeads were then washed several times with cold DI water. The crosslinker, hexamethylenediamine, was then added to the alginate microbeads dispersed in deionized water to give 1.5 equivalents per monosaccharide unit of alginate. After shaking at room temperature for 12 h, the covalently-crosslinked alginate microbeads were retrieved by washing with DI water several times. To generate macropores, CaCO3 microparticles were removed from the microbeads by dispersing them in a 50 mM EDTA solution for 2 h while under shaking. The resulting macroporous alginate microbeads loaded with Fe3O4 nanoparticles, designated as magnetic macroporous alginate microbeads, were collected


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after washing with PBS three times and dispersing in PBS. The swelling ratios of macroporous alginate microbeads were calculated by the following equation, Swelling ratio = (Ws-Wd)/Wd × 100% where Wd and Ws represent the weights of the freeze-dried microbeads and the microbeads at swelling equilibrium state, respectively. Adsorption of OVA and gold nanoparticles. Macroporous ferrogel microbeads were mixed with a 20 mg/mL OVA solution in acetate buffer, pH 4.5. The mixture was placed in a shaker at room temperature. The amount of OVA adsorbed was quantified by measuring the OVA remaining in the supernatant using a UV-Vis spectrometer (Multiskan GO, Thermo Scientific) at an absorbance of 280 nm. For gold nanoparticle loading, macroporous ferrogel microbeads were mixed with 13-nm citrate-stabilized gold nanoparticles dispersed in DI water (0.46 mg/mL) and the mixture was placed in a shaker at room temperature overnight. After the separation of the macroporous ferrogel microbeads, the concentration of the remaining gold nanoparticles in the supernatant was determined by measuring the absorption of the gold nanoparticles using a UVVis spectrometer. On-demand release by magnetic actuation. Five milliliters of a 5 mg/ml FITC-dextran (70,000 MW) solution was mixed with the magnetic macroporous ferrogel microbeads and incubated for an hour at room temperature. After incubation, the supernatant was removed and the microbeads were washed with PBS three times. For magnetic actuation, the bead sample dispersed in PBS was exposed to a repeated external magnetic field for one minute while the control sample was kept in PBS without the application of a magnetic field. The supernatants were collected at the desired time points and the absorbance of FITC in the supernatant was measured by a UV-Vis spectrometer to determine the amount of BSA-FITC released from the microbeads.



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In vitro drug delivery via magnetic actuation. 4T1 cells (2 × 104 cells well−1) were initially seeded in 24-well plates and cultured overnight. To prepare mitoxantrone-loaded ferrogel microbeads, 200 µg of mitoxantrone in 3 mL water was mixed with 2 mL of magnetic macroporous ferrogel microbeads and incubated for one day at room temperature to enable sufficient loading. The loading efficiency was checked by measuring the UV-Vis absorption of the supernatant. 250 µL of macroporous ferrogel microbeads loaded with 25 µg mitoxantrone were added to each well and subsequently actuated by a solid magnet for 15 min. The control group was aged after the addition of mitoxantrone-loaded ferrogel microbeads without magnetic actuation. The macroporous ferrogel microbeads were then removed from the cell culture media and the cells were cultured for 2 h. After a fresh culture media change, the cells were incubated for 24 h and then the cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay following the manufacturer’s instructions. Characterization and statistical analysis. The microbeads were observed under an optical microscope (Carl Zeiss, Primo Vert). The surface morphology of the freeze-dried microbeads was analyzed using a field emission scanning electron microscope (FE-SEM, JEOL, JSM6700F) The remaining Ca and Ba ions in the freeze-dried ferrogels microbeads were analyzed by energy dispersive X-ray spectrometry (EDS) attached on FE-SEM. All values were expressed in the form mean ± S.D. Differences between the groups were analyzed using a two-tailed Student’s ttest, and a P value of less than 0.05 was considered significant.


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ASSOCIATED CONTENT The Supporting Information [Supporting Figures S1-S4] is available free of charge via the ACS publications website at DOI: http://pubs.acs.org. Optical microscope images, EDS analysis, adsorption of protein and nanoparticles, and magnetic field simulation

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by grants funded by the National Research Foundation (NRF) under the Ministry of Science, ICT & Future Planning, Republic of Korea (2015R1A2A2A01005548, 2010-0027955), and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI17C0076).



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FIGURES

Scheme 1. Schematic representation of (a) preparation of injectable macroporous alginate ferrogel microbeads based on a templated method and (b) their application to magnetically actuated on-demand drug delivery.



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Figure 1. (a)–(f) Optical microscope images of the alginate-based macroporous hydrogel microbeads at each fabrication step: (a) initial Ca2+-crosslinked alginate microbeads embedded with CaCO3 microparticles and iron oxide nanoparticles (microbead size: 790 µm); (b) sizereduced microbeads after gradual solvent exchange with isopropanol (IPA) and subsequent Ba2+ crosslinking (microbead size: 550 µm); (c–f) covalently-crosslinked microbeads upon EDTA treatment for (c) 10 min (d) 30 min (e) 50 min, and (f) 1 h (microbead size: 750 µm); (g, h) High magnification optical microscope image of macroporous alginate microbeads at different magnifications; (i) Optical microscope image of non-macroporous Ca2+-crosslinked alginate microbeads.


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Figure 2. (a) Schematic of covalent crosslinking of alginate chains in hydrogel microbeads and after subsequent EDTA treatment; (b) Detailed covalent crosslinking mechanism between alginate polymer chains by CNBr activation at high pH and the subsequent reaction with hexamethylenediamine as a crosslinker. Optical microscope images of alginate hydrogel microbeads loaded with CaCO3 microparticles: (c) without covalent crosslinking and (d, e) with covalent crosslinking at a lower pH, (d), and at a higher pH, (e), over time during the EDTA treatment.



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Figure 3. Scanning electron microscope (SEM) images of (a) CaCO3 microparticles used as templates; (b) macroporous alginate microbeads prepared without applying the RsC process, showing collapsed and contracted structure upon lyophilization due to the low mechanical stability of hydrogel matrix, and (c, d) macroporous alginate microbeads prepared by the RsC process and subsequent EDTA treatment, showing maintained macropores and an overall microbead structure without severe deformation upon lyophilization. (b) and (d) are higher magnification images of (a) and (c), respectively.


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Figure 4. Photographic images of (a) non-macroporous and (b) macroporous ferrogel microbeads before and after exposure to an external magnetic field.



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Figure 5. (a) Dextran release from macroporous ferrogel microbeads without magnetic stimulation and under repeated magnetic stimulation. The magnetic field was applied for 1 min every 10 min; (b) Fluorescent image of fluorescently labeled ovalbumin released from macroporous ferrogel microbeads without magnetic stimulation or under magnetic stimulation; (c) Viability of cells incubated without macroporous ferrogel microbeads (control), and with macroporous ferrogel microbeads loaded with mitoxantrone with and without magnetic actuation; (d) Photographs of nude mouse subcutaneously injected with macroporous ferrogel microbeads through a needle (18 gauge) injection.


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