Magnetic Field Stimuli-Sensitive Drug Release Using a Magnetic


Dec 27, 2018 - A new stimulus-responsive drug delivery system using Fe3O4 nanoparticles coated with molecularly imprinted polymer (MIP) is reported...
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Magnetic Field Stimuli-Sensitive Drug Release Using a Magnetic Thermal Seed Coated with Thermal-Responsive Molecularly Imprinted Polymer Takuya Kubo,*,† Kaname Tachibana,† Toyohiro Naito,† Sadaatsu Mukai,‡ Kazunari Akiyoshi,‡ Jeyadevan Balachandran,§ and Koji Otsuka†

ACS Biomater. Sci. Eng. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/12/19. For personal use only.



Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan ‡ Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan § Department of Material Science, University of Shiga Prefecture, 2500 Hassaka-cho, Hikone City, 522-8533 Shiga Prefecture, Japan S Supporting Information *

ABSTRACT: A new stimulus-responsive drug delivery system using Fe3O4 nanoparticles coated with molecularly imprinted polymer (MIP) is reported. Magnetic thermal seeds (MTS) with their size controlled between 10 and 20 nm that could generate heat under an alternate current (AC) magnetic field were modified with a thermalresponsive MIP by grafting polymerization for effective release of an anticancer drug, methotrexate (MTX). The MIP-coated MTS showed the superparamagnetic property as well as the selective adsorption ability toward MTX, and 80% of MXT adsorbed on the MIP-coated MTS was stimulus released at 60 °C by cleaving hydrogen bonding in the recognition sites. Finally, the MTX release from the MTX-loaded MIP-coated MTS under an AC magnetic field within 10 min was successfully demonstrated. KEYWORDS: magnetic thermal seed, molecularly imprinted polymer, stimuli-sensitive drug release, AC magnetic field

1. INTRODUCTION

hybridization with magnetic nanoparticles is predominant.17−20 These studies have revealed the possibility of directing MIP-based DDS to the targeting area. In general, magnetite nanoparticles with an average diameter of about 20 nm exhibit superparamagnetic property and possess considerable saturation magnetization to be directed to specific parts of the body using a weak external magnetic field.21,22 Additionally, these magnetic nanoparticles interestingly generate heat under an alternate current (AC) magnetic field due to the relaxation of its magnetic moment. Moreover, the amount of heat generated is controllable by modulating the strength of the magnetic field and/or size of the nanoparticles.23,24 Consequently, the magnetite nanoparticles are employed as a magnetic thermal seed (MTS) in cancer therapy using magnetic hyperthermia.22,25 However, at present, the annihilation of cancer cells only by hyperthermia effect is considered difficult, and the concurrent use of anticancer drug treatment has been proposed. Thus, the development of novel DDS carrier that enables directivity toward the targeting area

Molecular imprinting technique, which enables creation of specific molecular recognition sites inside of a cross-linked polymer for selective adsorption of targeting compounds, has been widely studied in various fields.1−6 In recent studies, molecularly imprinted polymers (MIPs) have attracted more attention for stimulus-responsive capturing/releasing under external environmental conditions such as pH and temperature.7−11 Therefore, the most notable application of stimulusresponsive MIPs is as a carrier of a drug delivery system (DDS). As is well-known, a DDS provides an effective medicinal virtue with lower side effects by controlled release of drugs on significantly localized sites in the body.12−14 The two essential requirements of DDS are the directivity toward the targeting area and the controlled release of drugs. It is believed that MIPs are capable of meeting these requirements, and considerable interest has been shown in the development of MIPs-based new DDS carriers. However, typical MIPs are prepared with organic polymers, and the spontaneous transportation of these entities to affected parts cannot be achieved. Instead, MIPs hybridized with inorganic nanoparticles are considered very suitable.15,16 Briefly, a number of organic−inorganic hybrid nanomaterials has been studied; © XXXX American Chemical Society

Received: November 9, 2018 Accepted: December 27, 2018 Published: December 27, 2018 A

DOI: 10.1021/acsbiomaterials.8b01401 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Shizuoka, Japan) was used as a AC magnetic field generator, and H103NR (Kokusan Co. Ltd. Saitama, Japan) was used as a centrifuge. Zetasizer Nano ZSP (Malvern Panalytical, Malvern, UK) was used for dynamic light scattering (DLS) measurements. 2.3. Preparation of MIP-MTS Hybrid Particles. 2.3.1. Preparation of Magnetic Nanoparticles. The Fe3O4-based magnetic nanoparticles were prepared by the coprecipitation method as well as using the previous procedures reported by the authors.24 Briefly, iron(III) chloride hexahydrate of 2.16 g (8.0 mmol) and iron(II) chloride tetrahydrate of 0.97 g (4.9 mmol) were dissolved in 30 mL of pure water, and the mixture was treated by sonication. After nitrogen bubbling, the mixture was stirred at 700 rpm, and 0.74 M aqueous ammonia of 70 mL was dropped (15 drops/min). The Fe3O4 nanoparticles were collected by magnetic decantation and washed with pure water (3 times), and the collected particles were dispersed in methanol (MTS). 2.3.2. Modification of Vinyl Groups. The Fe3O4 nanoparticles dispersed in methanol (23 mg in 2.4 mL methanol) with 225 μL of pure water were treated by sonication. TEOS of 16.8 μL and 25% aqueous ammonia of 120 μL were added to the Fe3O4 nanoparticle dispersion, and then the mixture was stirred by 800 rpm at 40 °C for 2 h. The particles were collected by magnetic decantation. These procedures were repeated three times. After addition of methanol of 5 mL, the mixture was stirred at 60 °C for 6 h. The collected TEOS modified nanoparticles (TEOS-MTS) of 10 mg were dispersed in 1.5 mL of water/methanol (1/9), and 45.0 μL of acetic acid and 35.8 μL of VTMS were added. The mixture was heated to 60 °C and stirred at 800 rpm for 24 h. After magnetic decantation, the collected nanoparticles were washed with methanol (3 times) and dried at 50 °C in vacuum (VTMS-MTS) 2.3.3. MIP Coated MTS. According to our previous study,39 thermal-responsive MIP for MTX was modified onto the prepared VTMS-MTS. As shown in Table 1, folic acid was employed as a

and the controlled release of the drug is still strongly required. Only a few researchers previously reported the application of MTS using a typical thermoresponsive polymer, poly(Nisopropylacrylamide).26−29 Recently, a variety of magnetically triggered nanomaterials has been reported for new stimulus drug releasing.30−33 In these reports, the kind of MTS was employed for the trigger of temperature increase, and the sensitive drug release was achieved for certain encapsulated molecules. These are suitable for a general method enabling drug release of variety of molecules. As a further possibility, the hybridization of MIP and MTS is more attractive for controlled drug releasing toward a targeting molecule. Griffete et al. showed the possibility of the hybridization of MTS and MIP for DDS;34 however, longer period was needed for increase in temperature and drug release. Additionally, Chen et al. reported the magnetic lysozyme-imprinted nanoparticles and their releasing property due to near-infrared light.35 These reports strongly showed the possibility to employ MIP and MTS as a new concept for stimulus drug releasing. Hence, to clarify the possibility of using MTS and MIP for DDS, further investigations are strongly required. In this article, the concept for the development of a new DDS carrier hybridizing thermal-responsive MIP with Fe3O4 nanoparticle has been proposed and experimentally verified. The use of MIP as a carrier for an anticancer drug, methotrexate (MTX, see Figure S1)36−38 has been already demonstrated by the authors.39 In this previous study, we simply showed the possibility of a thermoresponsive releasing of MTX using a typical MIP consisted of only organic polymer. Therefore, to achieve a self-drug release, the additional functions for leading and increase temperature are necessary. Here, the hybridization procedures for the development of thermosensitive DDS (MIP-MTS) that generates heat and also releases drug simultaneously is reported. Then, the selective adsorption/desorption of MTX on the prepared MIP-MTX is evaluated. Finally, the release of MTX from the MIP-MTS under an AC magnetic field is also demonstrated.

Table 1. Components of the Prepared Hybrid Particles abbreviations

folic acid (mg)

MIP-MTS-1 NIP-MTS-1 MIP-MTS-2 NIP-MTS-2

3.9 − 3.9 −

DVB (μL)

MAA (μmol)

DMSO (mL)

60

2

84 167

initiator (mg)

VTMSMTS

2.9 10 5.9

2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. Dimethyl sulfoxide (DMSO), folic acid, methanol (HPLC grade), phosphoric acid, sodium dihydrogen phosphate dehydrate, sodium hydroxide (NaOH), sodium hydrogen carbonate (NaHCO3), ethanol, acetic acid, and tetraethyl orthosilicate (TEOS) were purchased from Nacalai Tesque Inc. (Kyoto, Japan). 2,2′-Azobis(2,4-dimethylvaleronitrile) (ADVN), methotrexate (MTX), iron(III) chloride hexahydrate, iron(II) chloride tetrahydrate, polyethylene glycol 4000 (PEG4000), Nphthaloyl-L-glutamic acid, trimethoprim, and 25% ammonia solution were purchased from Wako Pure Chemical Industries (Osaka, Japan). Divinylbenzene (DVB), methacrylic acid (MAA), and vinyltrimethoxysilane (VTMS) were purchased from Tokyo Chemical Industry (Tokyo, Japan). DVB and MAA were purified by distillation at 43 °C at 7.0 Torr and 53 °C by 24 Torr, respectively. Deionized water was obtained from a Milli-Q Direct-Q 3UV system (Merck Millipore, Tokyo, Japan). 2.2. Instruments. The high performance liquid chromatography system consisted of a pump (LC-10ADvp), a degasser (DGU-14AM), a column oven (CTO-10ACvp), and a photodiode array detector (SPD-M10Avp) (Shimadzu Co. Kyoto, Japan). Thermomixer C (Eppendorf AG. Hamburg, Germany) was used as a shaker; Digital Homogenizer HOM (AS ONE Co. Tokyo, Japan) was used as a mechanical stirrer, and TM-VSM 1230-HHH5 (Tamakawa Co., LTD, Sendai, Japan) was used as a vibrating sample magnetometer. Nicolet iS5 ATR (Thermo Fisher Scientific Inc. Waltham, MA, United States) was used as a FT-IR spectrometer; T162-5723A (Thamway Co. Ltd.,

pseudo template molecule, MAA as a functional monomer, and DVB as a cross-linker. Following the contents in Table 1 and Figure 1, VTMS-MTS was dispersed in the mixture of folic acid, MAA, DVB, and DMSO as a porogenic solvent. After sonication for 10 min, the mixture was stirred at ambient conditions for 1 h to construct the

Figure 1. Chemical structure of methotrexate and others. B

DOI: 10.1021/acsbiomaterials.8b01401 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering Scheme 1. Schematic Image of the Preparation of Particles and Thermoresponsive Releasing

Figure 2. TEM images of the MTS and the modified-MTSs. (top row) Lower magnitude; (bottom row) higher magnitude. complex of folic acid and functional monomers. Then, ADVN was added as a radical polymerization initiator, and the mixture was stirred at 800 rpm at 40 °C for 24 h to complete the surface modification of MIP layer onto VTMS-MTS. After polymerization, the particles were washed with acetic acid/methanol (1/9) to remove the template molecule (folic acid) and unreacted reagents. Finally, the particles were collected by magnetic decantation and dried under vacuum, and MIP-MTS were produced. Furthermore, the nonimprinted polymer coated MTS particles were also prepared by the same procedures without template molecules, and MIP-MTS were produced. For comparison with these hybrid particles, the typical MIP without any nanoparticles was also prepared by same composition of MIP-MTS-2 without MTS. The schematic image of the preparation of hybrid particles and its thermoresponsive molecular recognition is summarized in Scheme 1. 2.4. Adsorption and Desorption of MTX. MIP-MTS or NIPMTS particles were dispersed in 0.5−100 μM MTX (in DMSO/water = 6/4). Here, the concentration of the particles was adjusted to 5.0 mg/mL. The mixtures were stirred by 800 rpm at 25 °C for 24 h. After magnetic decantation, the concentrations of free MTX in supernatant were determined by HPLC analysis to estimate the amount of adsorbed MTX onto each particle. In addition, the collected MIP-MTS or NIP-MTS using a 50 μM MTX were washed with pure water (5 times) to remove the physically adsorbed MTX

and dried under vacuum for 48 h. The dried particles were redispersed in DMSO/water (6/4) of 5.0 mL and left at 60 or 25 °C for 24 h. After magnetic decantation, the free MTX was determined by HPLC to estimate the amount of released MXT. The HPLC conditions for determination of MTX are summarized as follows: column, YMC-Pack ODS-AM (150 mm × 4.6 mm i.d.) (YMC Co. Kyoto, Japan); mobile phase, (A) 10 mM phosphate buffer (pH 2.7), (B) methanol 20% B to 60% B for 6.0 min by linear gradient; temperature, 40 °C; detection, UV 300 nm. Calibration curve in HPLC analysis for MTX determination is indicated in Figure S1. The batch adsorption and desorption was considered by the following equation. Q = (Q 0 − C) × V /W

(1)

where Q is adsorption concentration of MTX on polymers (μmol/g), Q0 is MTX concentration of original solutions, C is MTX concentration after batch adsorption, V is volume of MTX solutions, and W is amount of polymers. 2.5. Evaluation of Magnetizing Properties. Magnetic properties of all the prepared particles were evaluated with a vibrating sample magnetometer. Furthermore, to confirm the heat generation under AC magnetic field, all the prepared particles were dispersed in a solvent and set at the center of handmade coil. After AC magnetic field was applied (frequency, 600 kHz; strength of magnetic field, 3.2 C

DOI: 10.1021/acsbiomaterials.8b01401 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering kA/m), the alteration of the temperature was evaluated, and the specific adsorption ratio (SAR) was estimated using the following equation. SAR = CS(mS /mi )(dT /dt )initial

confirmed the increase in the intensity of the peak corresponding to Si−O. This was considered due to the formation of additional Si−O through the construction of VTMS. Additionally, the absorptions based on vinyl groups were also recorded around 1400 cm−1 (C−H bending) and 1600 cm−1 (CC stretching). The above observations confirmed the effective modifications by TEOS and VTMS. In addition, the polymer coated nanoparticles were also analyzed by FT-IR as shown in Figure S2, and all the MIPMTS showed spectra similar to an authentic MIP, which was simply prepared without any nanoparticles and expressed as ncMIP (noncomposited MIP). According to these spectral results, the successful modification of MIP layer onto VTMSMTS by grafting polymerization was confirmed. Furthermore, TEM images and the appearances by magnetic decantation of these MIP-MTS are shown in Figures 2 and 3. As shown in TEM images, the TEOS and VTMS coated MTS were still dispersible in methanol. In contrast, the MIP coated magnetite nanoparticles were slightly aggregated. The polymer layer was gradually grown by increasing the amount of cross-linker. On the other hand, although the color change was clearly observed due to the increase in MIP content, all the MIP-MTS were still effectively collected by magnetic decantation. This confirmed the coating of MIPs onto the VTMS-MTS. To confirm the fraction of Fe3O4 particles and other layers, including TEOS, VTMS, and MIP, the magnetization of each sample was evaluated. The magnetization curve and saturation magnetization of each particle are summarized in Figure 4.

(2)

where CS is specific heat of the solvent, mS is amount of the solvent, mi is amount of magnetic particles, (dT/dt)initial is alteration of temperature per unit time

3. RESULTS AND DISCUSSION 3.1. Characterization of the Particles. We prepared the MIP-MTS by gradual modifications using TEOS and VTMS. The compositions of the layered MIPs and NIPs, which were prepared without any templates, are summarized in Table 1. TEM images in Figures 2 and 3 indicated that the

Figure 3. Physical appearance and magnetic decantation of MIPMTS. (left) Physical appearance and TEM image; (right) magnetic decantation; (upper) MTS; (middle) MIP-MTS-1; (lower) MIPMTS-2.

monodispersed Fe3O4 magnetic nanoparticles were successfully prepared. Additionally, the results of DLS observations for MTS and VTMS-MTS are summarized in Figure S3. Although DLS showed somewhat higher mean diameter that seemed to aggregate, by observation of these TEM images, the average diameter of the particles was estimated to be 11 nm, and its relative standard deviation was 20%. Figure 3 also shows the appearance during magnetic decantation. The results revealed that the nanoparticles were effectively dispersed in methanol and possessed the anticipated magnetization, which was revealed by the settling of the particles out by a permanent magnet. To confirm the surface modification of Fe3O4 particles, the FT-IR spectra of hybrid particles containing TEOS-MTS and VTMS-MTS were obtained, and the results are shown in Figure S2. In the case of TEOS-MTS, peaks corresponding to specific absorptions of Fe−O and Si−O were observed around 600 and 1100 cm−1, respectively, suggesting the successful TEOS modification. The comparison between the FT-IR profiles obtained for TEOS-MTS and VTMS-MTS

Figure 4. Magnetization curves for each nanoparticle.

Here, the weight fraction of Fe3O4 particles was estimated by comparing their saturation magnetization with the original Fe3O4 powder as shown in Table 2. According to Figure 4, original MTS and other three hybrid particles showed S shaped Table 2. Fraction of MTS Estimated from the Saturated Magnetization

MTS TEOS-MTS MIP-MTS-1 MIP-MTS-2 D

saturation magnetization (emu/g)

ratio of MTSs (%)

69.56 49.97 14.82 5.48

100 72 21 8 DOI: 10.1021/acsbiomaterials.8b01401 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ncMIP. Here, the compositions of MIP in MIP-MTS-2 was completely same as ncMIP, and the adsorption of MTX in original MTS was marginal. This can be due to different molecular recognition mechanisms caused by the hybridization with the nanoparticles (VTMS-MTS) and MIPs. Briefly, during the preparation of MIP-MTS, the template molecules first interacted with the remaining silanol groups of TEOS on VTMS-MTS, and then the polymerization was carried out with additional functional monomer, methacrylic acid (MAA). Therefore, the recognition sites were rigidly constructed on the surface of the nanoparticles. Consequently, both the amount and selectivity for MTX were dramatically increased in MIP-MTS. Furthermore, the adsorbed amount of MTX in MIP-MTS-2 was twice that of MIP-MTS-1, and the imprinting factor was over 5.0. These significant differences occurred simply due to difference in the amount of the MIP layer, while both NIP-MTS showed similar amount of MTX adsorption. Moreover, the adsorption selectivity for other related compounds was investigated to understand the selectivity for MTX and the molecular recognition mechanism in MIP-MTS. Usually, MIPs show cross selectivity for the structurally related compounds.40−44 Therefore, we investigated the adsorption selectivity for the compounds having structures with the similar moiety such as MTX, N-phthaloyl-L-glutamic acid, and trimethoprim (see Figure 1), which are analogues of glutamic acid and pteridine moiety, respectively. Batch adsorptions of these compounds for MIP-MTS-2 and NIP-MTS-2 were carried out, and the results are summarized in Figure S4. As expected, the imprinting factors for N-phthaloyl-L-glutamic acid and trimethoprim were 1.04 and 1.21, respectively. These results exhibited a slight enhancement in selective recognition of MIP for compounds structured with moieties similar to that of MTX. On the other hand, MIP-MTS-2 showed higher amount of MTX adsorption, and its imprinting factor was 5.04. Consequently, it is assumed that the significantly higher recognition selectivity of MTX in MIP-MTS-2 was due to three-dimensionally recognized multiple interaction. Additionally, to know the quantitative binding strength toward MTX in MIP-MTS, the isotherm adsorptions were evaluated. To evaluate the binding properties, we employed Langmuir

curves and disappearance of hysteresis, suggesting superparamagnetic behavior. As expected, the saturated magnetization gradually decreased with the increase in the thickness of MIP layer. 3.2. Adsorption Selectivity. The amounts of MTX adsorption toward MIP-MTS, ncMIP, and Fe3O4 (MTS) are summarized in Figure 5. According to our previous study,39

Figure 5. Adsorption of MTX on MIP/NIP-MTS-2. The amount of MTX adsorbed and imprinting factor (IF) of MIP-MTS-2 using 5 μM MTX solutions. IF was calculated from the adsorbed amount of MIP and NIP; IF = AMIP/ANIP (AMIP, adsorption amount on MIP; ANIP, adsorption amount on NIP). The batch adsorption and desorption were evaluated by using the following equation: Q = (Q0 − C) × V/ W, where Q is adsorption concentration of MTX on the nanoparticle (μmol g−1), Q0 is MTX concentration of original solutions, C is MTX concentration after batch adsorption, V is volume of MTX solutions, and W is amount of the nanoparticles.

selective adsorption of ncMIP was confirmed against ncNIP. Interestingly, the amount of adsorbed selectivity against the NIPs in each MIP-MTS was significantly higher than that of

Figure 6. Scatchard plots of MIP-MTS-2 and NIP-MTS-2. The Scatchard plots are prepared using the Langmuir model and the following equation: Cb/Cf = nKP − KCb, where Cb, Cf, n, and K are the amount of bounded MTX, the free concentration of MTX in solution after the equilibrium, the number of the binding sites, and the binding constant with MTX, respectively. E

DOI: 10.1021/acsbiomaterials.8b01401 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering model and Freundlich mode to estimate a few parameters.45−47 Although Freundlich mode showed better linearity as shown in Figure S5, Langmuir model showed the typical results due to the imprinting sites, which usually show bimodal recognition sites containing high and low affinity. The Scatchard plots of batch adsorption are shown in Figure 6, and the binding constants estimated by the Langmuir model are listed in Table S3. When NIP-MTS-2 was employed as adsorbent, lower binding constants (7.60 × 103 M−1) were estimated over a wide range of MTX concentrations. The adsorption behavior over the entire MTX concentrations could be explained using two binding constants. Similar behavior is observed in MIP studies;,48−52 in brief, the higher binding constant (1.18 × 105 M−1) of MIP-MTS-2 was 15 times that of NIP-MTS-2. The results clearly confirmed the existence of selective molecular recognition sites in MIP-MTS-2. 3.3. Releasing MTX under High Temperature. Figure 7 showed the amount and ratio of the desorbed MTX under

3.4. Release by AC Magnetic Field. Table 4 shows the temperature variation using magnetic nanoparticles under AC Table 4. Temperature Alterations on MIP-MTS-2 alteration of temperature (°C) MTS TEOS-MTS MIP-MTS-1 MIP-MTS-2

actual (°C)

theoretical (°C)

22.6 9.3 3.1 1.8

16.3 4.75 1.8

magnetic field. Here, the theoretical values of temperature alteration were calculated by using the amount of Fe3O4 particles. The actual temperature variations were slightly lower than the estimated theoretical values. This is assumed due to the heat loss caused by the modification of Fe3O4 particles by TEOS, VTMS, and MIPs. To confirm the heating phenomena of the Fe3O4 particles, the temperature variation against the concentration of the particles was measured, and the SAR values evaluated are summarized in Figure 8. Over the

Figure 7. Releasing of MTX on MIP/NIP-MTS-2. For the amount and ratio of released MTX from MIP-MTS-2, MTX was adsorbed on MIP- or NIP-MTS-2 with 50 μM MTX solution in advance, and then the amount of adsorbed MTX was estimated. After drying, the release was carried out in a MTX-free solvent at 25 or 60 °C for 10 or 60 min. Finally, the released MTX was estimated by determination of the concentration of MTX in supernatant.

Figure 8. Temperature alterations and SAR values on MTS.

concentration range considered, the theoretical linear relation between the MTS concentration and temperature variations was confirmed. On the other hand, as shown in Figure S6, when MIP-MTS-2 was employed for the similar evaluations, a linear relation was not observed, especially at higher concentrations. At lower concentrations of MIP-MTS, a linear relationship was confirmed, however, and with the increase in concentration, the temperature remained almost unchanged. As shown in the TEM image of MIP-MTS, the hybrid MIP particles were not monodispersed due to the aggregated morphology. Accordingly, it is assumed that the aggregation of each particle inhibited the theoretical heat dissipation. Therefore, to obtain the theoretical temperature increase in MIP-MTS under AC magnetic field, the concentration should be under 25 mg/mL. Considering the results related to selective adsorption/ desorption of MTX and heating function in MIP-MTS, finally the drug release by simply applying AC magnetic field was demonstrated. The released MTX was estimated using varying concentrations of MTX-adsorbed MIP-MTS-2. The results are summarized in Figure 9. The difference in the amount of MTX

variety of temperatures and soaking time. According to these results, increasing desorption ratios were observed in both MIP and NIP at higher temperatures. However, the desorption ratio of NIP was higher than that of MIP. It is inferred that MTX adsorbed on NIP weakly through hydrogen bonding with randomly arranged MAA in NIP, and 60 °C was enough to cleave most of the interactions. On the other hand, around 20% MTX remained in MIP-MTS-2 even at 60 °C, which indicated the existence of significantly strong recognition sites in MIP. In the case of soaking time, MTX was completely released within 10 min at 60 °C, while the releasing was not completed at 25 °C even after 60 min. These results indicated that the multiple interactions due to through hydrogen bonding could be cleaved at higher temperature even in the case of MIP. Considering the above results, including the adsorption tests, the amount of MTX released was much higher in MIP-MTS-2 and is highly anticipated to be used for the effective drug release by modulating the temperature. F

DOI: 10.1021/acsbiomaterials.8b01401 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 9. Releasing MTX from MIP-MTS-2 by AC magnetic field. (a) Amount and (b) ratio of released MTX from MIP-MTS-2 with/without AC magnetic field. The ratio was estimated from the adsorbed MTX on MIP-MTS-2 in advance.



released was clearly confirmed under the presence and absence of AC magnetic field. The results strongly suggested that the core Fe3O4 particles were heated by magnetic field, and then the adsorbed MTX was effectively cleaved from the MIP layer. On the other hand, the ratio of MTX released was almost same over 10 mg/mL of MIP-MTS-2 (Figure 9b), the reason being that the heating plateaued at higher concentration of MIPMTS, as observed in the heating test in Figure S6. Altogether, the power of magnetic field was limited to make the particles heating in this case. Further effective release was achieved by using higher AC magnetic field. Consequently, the possibility of the concept using thermal-responsive MIP and magnetic thermal seed for efficient drug release as a new DDS was successfully demonstrated.

AUTHOR INFORMATION

Corresponding Author

*Tel: +81-75-383-2448; Fax: +81-75-383-2450; E-mail: [email protected] ORCID

Takuya Kubo: 0000-0002-9274-3295 Sadaatsu Mukai: 0000-0001-9909-9116 Koji Otsuka: 0000-0003-1088-0569 Author Contributions

T.K., K.A., B.J., and K.O. designed and conducted experiments for surface modification and adsorption/releasing procedures. K.T., T.N., and S.M. performed the characterizations and analyzed data. Notes

The authors declare no competing financial interest.

5. CONCLUSION In conclusion, a novel DDS concept using the hybrid material composed of magnetic-field stimuli-sensitive seed (MTS) and a thermal-responsive MIP was experimentally verified as follows. The targeting drug, MTX, was selectively adsorbed on MIP-coated MTS through hydrogen bonding and desorbed by simply controlling the temperature by applying AC magnetic field using the heat generating property of MTS that converts magnetic energy into thermal energy. The proposed concept can be effectively used for a stimulus DDS by further optimizations containing the materials of MIPs and the strength of magnetic field.





ACKNOWLEDGMENTS This research was partly supported by the Grant-in Aid for Scientific Research (Grants 25620111 and 15K13756) from the Japan Society for the Promotion of Science, an Environment Research and Technology Development Fund (5-1552) from the Ministry of the Environment, Japan, and JST CREST Grant JPMJCR17H2, Japan.



REFERENCES

(1) Ansari, S.; Karimi, M. Novel developments and trends of analytical methods for drug analysis in biological and environmental samples by molecularly imprinted polymers. TrAC, Trends Anal. Chem. 2017, 89, 146−62. (2) Ashley, J.; Shahbazi, M. A.; Kant, K.; Chidambara, V. A.; Wolff, A.; Bang, D. D.; Sun, Y. Molecularly imprinted polymers for sample preparation and biosensing in food analysis: Progress and perspectives. Biosens. Bioelectron. 2017, 91, 606−15. (3) Bazin, I.; Tria, S. A.; Hayat, A.; Marty, J. L. New biorecognition molecules in biosensors for the detection of toxins. Biosens. Bioelectron. 2017, 87, 285−98. (4) Speltini, A.; Scalabrini, A.; Maraschi, F.; Sturini, M.; Profumo, A. Newest applications of molecularly imprinted polymers for extraction

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b01401. Calibration curve for MTX in HPLC, FT-IR spectra, DLS of the prepared nanoparticles, and temperature alterations by MIP-MTS-2 (PDF) G

DOI: 10.1021/acsbiomaterials.8b01401 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

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DOI: 10.1021/acsbiomaterials.8b01401 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.8b01401 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX