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Lili Chen, [Nanami Fujisawa](https://orcid.org/0000-0002-8894-1790), Masato Takanohashi, [Mitsuhiro Ebara](https://orcid.org/0000-0002-7906-0350)

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[An injectable hyperthermic nanofiber mesh with switchable drug release to stimulate chemotherapy potency](https://mdr.nims.go.jp/datasets/8b762a1d-33f5-48dd-9799-38df793b505e)

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fbioe-2022-1046147 1..11An injectable hyperthermicnanofiber mesh with switchabledrug release to stimulatechemotherapy potencyLili Chen1, Nanami Fujisawa1,2, Masato Takanohashi1 andMitsuhiro Ebara1,2,3*1Research Center for Functional Materials, National Institute for Materials Science (NIMS), Tsukuba,Japan, 2Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan,3Department of Materials Science and Technology, Tokyo University of Science, Tokyo, JapanWe developed a smart nanofiber mesh (SNM) with anticancer abilities as well asinjectability and fast recovery from irregular to non-compressible shapes. Themesh can be injected at the tumor site to modulate and control anticancereffects by loading the chemotherapeutic drug, paclitaxel (PTX), as well asmagnetic nanoparticles (MNPs). The storage modulus of the mesh decreaseswhen applied with a certain shear strain, and the mesh can pass through a 14-gauge needle. Moreover, the fibrous morphology is maintained even afterinjection. In heat-generation measurements, the mesh achieved an effectivetemperature of mild hyperthermia (41–43°C) within 5 min of exposure toalternating magnetic field (AMF) irradiation. An electrospinning method wasemployed to fabricate the mesh using a copolymer of N-isopropylacrylamide(NIPAAm) and N-hydroxyethyl acrylamide (HMAAm), whose phase transitiontemperature was adjusted to a mildly hyperthermic temperature range.Pplyvinyl alcohol (PVA) was also incorporated to add shear-thinning propertyto the interactions between polymer chains derived from hydrogen bonding,The “on-off” switchable release of PTX from themesh was detected by the drugrelease test. Approximately 73% of loaded PTXwas released from themesh aftereight cycles, whereas only a tiny amount of PTXwas released during the coolingphase. Furthermore, hyperthermia combined with chemotherapy afterexposure to an AMF showed significantly reduced cancer cell survivalcompared to the control group. Subsequent investigations have proven thata new injectable local hyperthermia chemotherapy platform could bedeveloped for cancer treatment using this SNM.KEYWORDShyperthermia, injectable nanofiber, chemotherapy, magnetic nanoparticles,temperature-responsive polymersOPEN ACCESSEDITED BYLing Wang,Southern Medical University, ChinaREVIEWED BYJi-Huan He,Soochow University, ChinaTheodora Krasia-Christoforou,University of Cyprus, Cyprus*CORRESPONDENCEMitsuhiro Ebara,EBARA.Mitsuhiro@nims.go.jpSPECIALTY SECTIONThis article was submitted toBiomaterials,a section of the journalFrontiers in Bioengineering andBiotechnologyRECEIVED 16 September 2022ACCEPTED 14 October 2022PUBLISHED 03 November 2022CITATIONChen L, Fujisawa N, Takanohashi M andEbara M (2022), An injectablehyperthermic nanofiber mesh withswitchable drug release to stimulatechemotherapy potency.Front. Bioeng. Biotechnol. 10:1046147.doi: 10.3389/fbioe.2022.1046147COPYRIGHT© 2022 Chen, Fujisawa, Takanohashiand Ebara. This is an open-access articledistributed under the terms of theCreative Commons Attribution License(CC BY). The use, distribution orreproduction in other forums ispermitted, provided the originalauthor(s) and the copyright owner(s) arecredited and that the originalpublication in this journal is cited, inaccordance with accepted academicpractice. No use, distribution orreproduction is permitted which doesnot comply with these terms.Frontiers in Bioengineering and Biotechnology frontiersin.org01TYPE Original ResearchPUBLISHED 03 November 2022DOI 10.3389/fbioe.2022.1046147https://www.frontiersin.org/articles/10.3389/fbioe.2022.1046147/fullhttps://www.frontiersin.org/articles/10.3389/fbioe.2022.1046147/fullhttps://www.frontiersin.org/articles/10.3389/fbioe.2022.1046147/fullhttps://www.frontiersin.org/articles/10.3389/fbioe.2022.1046147/fullhttps://crossmark.crossref.org/dialog/?doi=10.3389/fbioe.2022.1046147&domain=pdf&date_stamp=2022-11-03mailto:EBARA.Mitsuhiro@nims.go.jphttps://doi.org/10.3389/fbioe.2022.1046147https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://www.frontiersin.org/journals/bioengineering-and-biotechnologyhttps://www.frontiersin.orghttps://www.frontiersin.org/journals/bioengineering-and-biotechnologyhttps://www.frontiersin.org/journals/bioengineering-and-biotechnology#editorial-boardhttps://www.frontiersin.org/journals/bioengineering-and-biotechnology#editorial-boardhttps://doi.org/10.3389/fbioe.2022.1046147IntroductionDrug delivery systems (DDSs) are being widely investigatedas technologies to reduce the effects of drugs on normal cellswhile specifically targeting cancer cells (Parisi et al., 2014;Sanadgol and Wackerlig, 2020). In a DDS, the drugdistribution and concentration are controlled spatiotemporallyto reduce side effects and improve drug efficacy by administeringonly the necessary and sufficient amount of the drug. In the studyof DDSs in the treatment of cancer, the enhanced permeationand retention (EPR) effect discovered by Maeda et al. has oftenbeen applied (Maeda, 2010). The EPR effect refers to the fact thatneovascularization, which occurs rapidly around canceroustumors, has an incomplete structure with gaps between thevascular endothelial cells. By leaking into the interstitialtissues, nanoparticles with diameters of 100 nm or less areeasily delivered to the tumor sites. A significant benefit of thiseffect is that it allows selective drug delivery to cancerous tumors(Maeda and Islam, 2020; Subhan et al., 2021). However, due tothe uncontrollable nature of passive targeting, there are still somechallenges to making EPR effect in clinical application.The concept of a reservoir formulation has attractedattention as a locally controllable DDS from a differentperspective than the EPR effect. A reservoir formulation is adrug-loaded formulation that is implanted in the patient’s bodyso that the drug can be released into the body at any time. Theformulation is implanted under the skin or in an affected area ofthe patient’s body through surgery involving incision andremoval (Sapino et al., 2019; Fayzullin et al., 2021); the aimhere is to administer the necessary and sufficient amount of thedrug at the appropriate time. The drug reservoir is often locatednear the affected area, allowing for physical space and temporalcontrol through the timing of drug administration from thereservoir. Reservoir-based formulations have been used forpatients who require long-term care and continuous drugadministration, such as those with hormonal imbalances orosteoporosis (Villarruel Mendoza et al., 2020), patientsundergoing chemotherapy supported by subcutaneous portimplantation, and diabetic patients (Anselmo and Mitragotri,2014; Sanjay et al., 2018; Li W et al., 2022) using insulin pumptherapy. From these clinical experiences, it is clear that reservoir-based formulations can improve the quality of life by preventingmissed or lost medications and reducing the physical and mentalburden of patients due to long-term drug dependency. Therefore,we developed and studied a smart nanofiber mesh (SNM) as anew cancer treatment platform that enables active targeting ofcancerous tumors by focusing on the concept of reservoir-basedformulation delivery.SNM is a type of nanofiber with “smart” properties, based ontheir physical or chemical properties such as “stimuli-responsive”and “environmental-sensitive”, which for such applications as“on-off” switchable control of swelling/deswelling and adhesionbehavior (Wang et al., 2019; Wang et al., 2022). This material canbe produced by electrospinning through a single needle(Khodadadi et al., 2020), coaxial electrospinning (Peng et al.,2021; Li J et al., 2022), or bubble spinning (He et al., 2022; Qianand He, 2022). The SNM is surgically implanted near the affectedarea and can effectively treat diseases through active targetingand long-term release of encapsulated drugs. As an example, anSNM targeted at carpal tunnel syndrome encapsulates aconventional therapeutic drug for carpal tunnel syndrome in ananofiber mesh composed of polycaprolactone. The SNM hasbeen successfully applied to stimulate the regeneration ofperipheral nerves, which is not possible with a singletherapeutic drug and is currently under clinical trials inhumans (Grinsell and Keating, 2014; Hoyng et al., 2015).In addition, a SNM for cancer therapy was developed usingpoly (N-isopropylacrylamide-co-N-hydroxymethyl acrylamide)(P(NIPAAm-co-HMAAm)), a temperature-responsive polymer,as the substrate and magnetic nanoparticles (MNPs) as theheating elements. HMAAm was used to provide the chemicalcrosslinking points as well as to adjust the phase transitiontemperature around hyperthermic temperature. The SNM wasdesigned to induce phase transition of P(NIPAAm-co-HMAAm)by heat generation from the MNPs, thus enabling acceleratedrelease of the drug. Furthermore, by maintaining the heatgeneration temperature around 42–43°C, we were able tocombine hyperthermia, a kind of heat-based cancer therapy,with chemotherapy by releasing the encapsulated drug. We aimto develop aminimally invasive therapy with fewer side effects onnormal cells through localized combined hyperthermia/chemotherapy.As a method of providing injectability, we considered usingthe shear-thinning property in the SNM. The shear-thinningproperty is a phenomenon by which a material becomes fluidunder an applied shear stress. This is known to occur when theshear stress increases the mobility of the molecules by breakingthe intermolecular interactions, orienting molecules along thestress direction, and removing entanglements between molecules(Dufresne and Castaño, 2017; Sadasivuni et al., 2019). Therefore,we decided to add polyvinyl alcohol (PVA) to the substrate of theSNM to control the interactions between the methylol group ofP(NIPAAm-co-HMAAm) and hydroxy group of PVA derivedfrom hydrogen bonding (Cho et al., 2010; Nuruddin et al., 2021).This study was aimed at applying the SNM to the affectedarea by injection without surgery. The injectable SNM not onlyenables minimally invasive implantation in the patient’s body butalso has the advantage of improved handling performance. Aminimally invasive SNM based on the P(NIPAAm-co-HMAAm)and PVA was developed for application by injection. This SNMresponds to an alternating magnetic field (AMF) owing to theloaded MNPs and shows temperature-responsive drug releasebehavior (Scheme 1). Therefore, the well-defined multifacetedplatform represents an appreciable SNM for efficient andminimally invasive distinctive therapy to the tumor cells,which is important in the validation of strategic pursuit ofFrontiers in Bioengineering and Biotechnology frontiersin.org02Chen et al. 10.3389/fbioe.2022.1046147https://www.frontiersin.org/journals/bioengineering-and-biotechnologyhttps://www.frontiersin.orghttps://doi.org/10.3389/fbioe.2022.1046147promoted combination therapeutic outcomes throughhyperthermia and chemotherapeutics.Results and discussionFabrication of smart nanofiber meshAs shown in Scheme 2, NIPAAm and HMAAm wererandomly copolymerized to form P (NIPAAm-co-HMAAm).In this study, a copolymer with a relatively high molecular weight(Mw 50 k~) was synthesized. As a result of insufficient molecularchain entanglement during electrospinning, the lower molecularweight may cause beads or particles to form. As molecular weightincreases, fiber morphology changes from beads to fine fibers.According to our previous report (Oroumei et al., 2015;Ahmadian et al., 2021), PNIPAAm with Mn = 10 k forms abead-like structure at 1.0–10 w/v%. With PNIPAAm with arelatively higher molecular weight, fibers were electrospun ona lower solution concentration of 1.0–3.0%.To adjust the shear-thinning property of SNM to achieveinjectability, the hydrophilic ability will be introduced. Herein,the fiber fabrication conditions were optimized as follows: 25-gauge needle, 20 wt% polymer concentration, three differentamounts (0, 5, 10 wt%) of PVA, 20 kV of applied voltage, anda 13 cm gap between the collector and the needle. A comparisonof the isopropyl group of the NIPAAm and the methylol group ofHMAAm by 1H NMR determined that the HMAAm content inthe copolymer was 20 mol%. Approximately 41–43°C wasselected as a mild-hyperthermia temperature for thecopolymer’s phase transition. The corresponding phasetransition temperature value was 42.1°C.Characterization of smart nanofiber meshIn this study, poly (NIPAAm-co-HMAAm)/PVA nanofiberswith different amounts of PVA were fabricated through blendelectrospinning. Figure 1A shows the optical microscopy andSEM images of a variety of electrospun nanofiber meshes afterthermal crosslinking. As shown in Figure 1A, the nanofiberbefore crosslinked were bead-free, smooth and uniform fiberswere formed with an average diameter of 1,120 ± 231, 640 ± 200,608 ± 190 nm, respectively, regardless of PVA content. It ismaybe the introduced of HMAAm units is to crosslink polymerchains, and this induced the stability of the meshes in aqueousmedium (Kim and Matsunaga, 2017; Niiyama et al., 2018; Zhaet al., 2021). Moreover, after thermal crosslinking, the nanofibersmaintained nanofibrous structures and the average diameter was1,111 ± 110, 509 ± 118, 547 ± 250 nm, respectively (Figure 1B).These results suggest that thermal crosslinking occurs onlywithin the fibers and does not contribute to inter-fiber adhesion.Furthermore, it has been confirmed that the nanofiber poly(NIPAAm-co-HMAAm)/PVA has a crosslinked structure by thedisappearance of peaks corresponding to methylol groups in theattenuated total reflection Fourier-transform infraredSCHEME 1Design of a smart injectable hyperthermia nanofiber system with MNPs dispersed in temperature-responsive polymers. The nanofibers alsoincorporate an anticancer drug. With the device signal (AMF) turned “on” to activate the MNPs in the nanofibers, the MNPs generate heat to collapsethe polymer network in the nanofiber, thus allowing “on-off” release of the drug. Following hyperthermia and chemotherapy, the heat and drugrelease induce apoptosis of the cancerous cells.Frontiers in Bioengineering and Biotechnology frontiersin.org03Chen et al. 10.3389/fbioe.2022.1046147https://www.frontiersin.org/journals/bioengineering-and-biotechnologyhttps://www.frontiersin.orghttps://doi.org/10.3389/fbioe.2022.1046147spectroscopy (ATR-FTIR) spectra (Figure 1C). The peaks at1,650 and 1,550 cm−1 were assigned to amide I (C=Ostretching) and amide II (N–H bending) of the copolymer,respectively. The broad absorption band observed around3,430 cm−1 was assigned to the N–H stretching of amidegroups in the copolymer. The peaks at 1,370–1,390 and2,980 cm−1 were assigned to the respective stretching modes ofNIPAAm’s–CH(CH3)2 and –(CH3)2 groups. The peaks at 1,050,1,230, and 3,300 cm−1 were assigned to the C–O–H stretching,C–O–H bending, and O–H stretching modes in HMAAm,respectively (Niiyama et al., 2018; Wei et al., 2022). Withincreasing thermal treatment time, the signal intensity of themethylol groups gradually decreased. It was confirmed that thesignals completely disappeared and the thermal crosslinking wascompleted in 6 h.Shear thinning properties of smartnanofiber meshIn recent years, electrospun nanofibers have made rapidadvances in cancer therapy due to their high drug loadingrates, high specific surfaces, and good drug release profiles.However, drug-loaded nanofibers are typically appliedexternally or injected through fibrous membranes to treatcancer, resulting in reduced complicated surgical operations,drug utilization, and secondary damage. For the purpose ofsolving these bottlenecks, injectable electrospun nanofibers(PNIPHM_PVA0, PNIPHM_PVA5, and PNIPHM_PVA10)thermally crosslinked with different ratios of PVA werefabricated. For investigating the injectability of the nanofibermeshes, the strain amplitude-dependent dynamic viscoelasticity,shear rate dependent viscosity, and continuous-step strainmeasurement were performed.The loss modulus of each SNM exceeded the storage moduluswhen the strain is applied above a certain threshold value(Figure 2A). The strain value at the point where the lossmodulus first exceeded the storage modulus was 379% forPNIPHM_PVA0, while it was 200% for PNIPHM_PVA5 andPNIPHM_PVA10. This result confirms the increase offlowability with the increase rate of PVA. Furthermore, theresults of shear rate dependent viscosity measurement wereshown in Figure 2B. The shear-thinning property of SNMwith any PVA content decreases with an increasing shear rate.The shear-thinning property of SNMs was confirmed. Inaddition, it can be noted that for all types of SNM, thestorage modulus was higher when the shear strain was 1%and the loss modulus was higher when the shear strain was1,000% in Figure 2C. This indicates that the mesh becomes fluidonly when shear strain is applied and recovers its shape when thestrain is removed. Moreover, at 1% shear strain, the storagemodulus increased with time. This indicates that the nanofibermesh absorbs water from the surrounding area after beingsubjected to strong shear force, and gradually recovers itsshape by swelling again. The storage modulus at thebeginning of the measurement (0 s) was set to 100%, and theshape recovery rate was calculated by comparing the storagemodulus at 120, 300, 480, 660, and 780 s. The shape recoveryrates were 103, 97, 78, 78, 109% for PNIPHM_PVA0, 128, 67, 55,49, 38% for PNIPHM_PVA5, and 113, 50, 23, 16, 13% forPNIPHM_PVA10. It was found that the shape recovery rateworsened with the increasing PVA addition rate. A possibleexplanation is that the addition of PVA, a crystalline polymer,increases the crystallinity of the nanofiber mesh, which in turnincreases the mechanical strength and decreases flexibility (Fariset al., 2021; Liang et al., 2021; Zhou et al., 2022). On the basis ofthose results, this composition (PNIPHM_PVA5) was selectedfor further study.SCHEME 2Diagrammatic illustration of the SNM with poly (NIPAAm-co-HMAAm)/PVA.Frontiers in Bioengineering and Biotechnology frontiersin.org04Chen et al. 10.3389/fbioe.2022.1046147https://www.frontiersin.org/journals/bioengineering-and-biotechnologyhttps://www.frontiersin.orghttps://doi.org/10.3389/fbioe.2022.1046147AMF-responsive heat generationThermal therapy known as magnetic hyperthermia involvesheating tumors using magnetic nanoparticles and applying anAMF to create heat (Albarqi et al., 2020; Gavilan et al., 2021).High temperatures (42–45°C)can kill cancer cells, as well act as asensitizer to enhance the effects of chemotherapy (Ahmed K,2012). As a result of its excellent ability to penetrate tissues, AMFcan precisely treat deep tumors in organs (Hayashi et al., 2016).In this regard, it is crucial to determine the heating potential ofMNPs within nanofiber meshes exposed to AMF. Generally,magnetic fields affect tissues and organ systems in certain ways,such as causing induced eddy currents in tissues, resulting innecrosis or carbonization in healthy tissues. Thereby, magneticfield forces are restricted. The AMF safety frequency threshold is100–300 kHz in clinical applications (Mamiya et al., 2017; Chenet al., 2021). In this study, an AMF of 281 kHz was used and itsintensity is relevant for clinical applications.To investigate the responses of the MNPs-loaded nanofibersto a magnetic field, Figure 3A shows the heating profiles of theSNMs with 30 wt% MNPs loading with AMF switching every360 s (on for 300 s and off for 60 s). The temperature of the SNMincreased from 23.7°C to 42.5°C caused by the irradiation of281 kHz AMF and dropped immediately to the startingtemperature once the irradiation was turned off. Furthermore,in Figure 3B, it shows the time-dependent temperature changesof the SNMs with different MNPs loading rates under AMFirradiation for 360 s. The temperature of each test sample sharplyincreased immediately after AMF irradiation, reaching a plateauwithin 30 s at 33.4°C, 42.1°C, and 47.3°C in meshes with 10 wt%,20 wt%, and 30 wt% MNPs loading, respectively. The resultindicated that the heating profiles are dependent on theFIGURE 1(A) SEM image and (B) the average diameters of the nanofibers: Poly (NIPAAm- c o-HMAAm) pre-crosslinked with PVA, poly (NIPAAm- coHMAAm) post-crosslinkedwith PVA and poly (NIPAAm- c o-HMAAm) crosslinkedwith PVA after injection. (C)Change in 1HNMR spectra of the poly(NIPAAm- c o-HMAAm)/PVA processed with different heating times.Frontiers in Bioengineering and Biotechnology frontiersin.org05Chen et al. 10.3389/fbioe.2022.1046147https://www.frontiersin.org/journals/bioengineering-and-biotechnologyhttps://www.frontiersin.orghttps://doi.org/10.3389/fbioe.2022.1046147concentration of MNPs loading, which will make the heatingability of the SNM more controllable. Based on these results, theSNM with 30% (9 mg) of MNPs was chosen for furtherinvestigation.“On-off” switchable drug releaseControllable drug release is an important evaluationcriterion for SNM. Next, the nanofiber meshes containingPTX, MNPs and PVA were used to verify the drug releasebehavior from nanofiber. NIPAAm-co-HMAAm polymer issoluble in aqueous media below LCST and dehydrates rapidlyabove LCST. Therefore, PTX is released from the polymeralong with the heating of MNPs induced by AMF. Figure 4shows the switching from “on” to “off” AMF applications onPTX release from nanofibers. It is worth noting thatapproximately 16% of loaded PTX were released in the firstheating process. However, the release was stopped aftercooling to room temperature, and restarted release duringthe second heating. It is demonstrated that the release of thePTX was accelerated by AMF irradiation and was stopped atroom temperature. Interestingly, with the increase of theswitching cycle number, the cumulative release of PTX alsoincreased significantly, and showed a switching cycle-dependent relationship. The release of the PTX cumulativeamount was over 70% within eight cycles. Owing to the highspecific surface area of nanofibers, their sensitivity to externalstimuli is greater than that of bulk materials (Lin et al., 2013).Thus, we took advantage of the nanometric effects ofnanofibers for the “on-off” switchable release of drugs.From this regard, these results in Figure 4 indicate that theproposed SNM system allows the control of switchable drugrelease by simply switching the AMF “on” and “off,” and thiscontrolled release SNM may be very promising for cancertherapeutics in the future.FIGURE 2(A) Result of strain amplitude dependent dynamic viscoelasticity measurement, (B) Result of shear rate dependent viscosity measurement, and(C) Result of continuous-step strain measurement (n = 3, mean ± SD).Frontiers in Bioengineering and Biotechnology frontiersin.org06Chen et al. 10.3389/fbioe.2022.1046147https://www.frontiersin.org/journals/bioengineering-and-biotechnologyhttps://www.frontiersin.orghttps://doi.org/10.3389/fbioe.2022.1046147Anti-tumor effectHybrid nanoarchitectures with magnetic nanoparticles havebeen used for hyperthermic cancer cell therapy in recent years(Lin et al., 2013). However, due to their nano dimensionalproperties, these hybrids cannot be directly applied to livingmatter, as they may lead to toxic side effects. Interestingly, one ofthe advantages of the SNM in this study is that a nanofiber as adressing can be manipulated and injected directly into the tumorregion. In this regard, the anti-tumor effects of the SNMs basedon hyperthermia and the chemotherapeutic effects of the MNPs/PTX@SNM was investigated using SKOV3 cells.Before co-culturing with nanofiber mesh the cell viability ofSKOV3 cells treated with PTX was assessed by the alamar Blueassay. A culture medium containing PTX was used to culture theSKOV3 cells at 0, 0.001, 0.1, 1.0, and 10 μg ml−1 concentration for24 h. As shown in Figure 5A, cell viability decreases as the drugconcentration increases, and IC50 of SKOV3 at 1.66 μg ml−1.Furthermore, the anti-tumor effects of the SNMs were furtherevaluated using SKOV3 cells. The cells were co-cultured with theSNMs for 24 h And the cells were exposed to AMF once every5 min and then at room temperature for 5 min for each cycle. Asdemonstrated in Figure 5B, the blank SNM under the AMFmaintained cell viability higher than 95%, indicating that theblank nanofiber mesh and magnetic field was non-toxic againstSKOV3 cells. On the other hand, with the SNM exposure toAMF, the cell viability of SNM loaded with MNPs alone (MNPs-SNM) and PTX alone (PTX@SNM) decreased graduallycompared to the control group (medium only). This may bedue to the effect of hyperthermia generated by MNPs-loadedSNM exposed to AMF and chemotherapy effect resulting fromPTX release from surface of nanofiber. Moreover, the cellsviability in the presence of MNPs and PTX co-loadednanofibers group was 60.45 ± 6.72. This result may be due tofew PTX being released from the surface of nanofibers withoutAMF application (“off” state). This result corresponds well to thein vitro drug release study (Figure 4). It can be noted that, the cellviability of MNPs/PTX@SNM with the magnetic field was only11.23% ± 5.01, because of the combined effect of hyperthermiawith PTX released caused by heating. In sum, the findingsdemonstrate that MNPs and PTX@SNM exposed to AMFgreatly increase cytotoxicity in SKOV3 cells. In light of this,this platform may be developed into an implantable deliverysystem for treating ovarian cancer.ExperimentalMaterialsDimethylformamide (DMF), 2′2-Azobis (isobutyronitrile)(AIBN) and N-Isopropylacrylamide (NIPAAm) werepurchased from Fujifilm Wako Pure Chemical CorporationFIGURE 3(A) Infrared thermal images of MNPs-loaded SNM with “on” or “off” AMF irradiation. (B) Heating profiles of MNPs@SNM with different MNPscontents during AMF application at different times.FIGURE 4Drug release behavior from SNM containing PTX (1.0 wt%),MNPs (0 or 20 wt%) and PVA (5 wt%) at different cycles of AFM.Frontiers in Bioengineering and Biotechnology frontiersin.org07Chen et al. 10.3389/fbioe.2022.1046147https://www.frontiersin.org/journals/bioengineering-and-biotechnologyhttps://www.frontiersin.orghttps://doi.org/10.3389/fbioe.2022.1046147(Osaka, Japan). The following products were purchased fromTokyo Chemical Industry Co., Ltd. (Tokyo, Japan):N-Hydroxymethylacrylamide (HMAAm), 1,1,1,3,3,3-Hexafluoro-2-propanol, and Paclitaxel (PTX). We obtainedferrofluid made of iron oxide (III) (10 nm particle size) fromFerrotec Holdings Corporation (Tokyo, Japan). Fetal bovineserum (FBS) was purchased from Tocris Bioscience Inc.(Minneapolis, MN, United States). McCoy’s 5 A MediumModified, content sodium bicarbonate, free of L-glutamine,liquid, and sterile-filtered for cell culture was purchased fromSigma-Aldrich Japan (Tokyo, Japan). We obtained the AlamarBlue reagent from TREK Diagnostic Systems (Cleveland, OH,United States). The American Type Culture Collection(Manassas, VA, United States) provided SKOV3 (humanovarian cancer cell line: adenocarcinoma).Synthesis and characterization ofP(NIPAAm-co-HMAAm)According to the previous description, NIPAAm andHMAAm were copolymerized. Briefly, 80 mol% NIPAAm wasdissolved in 20 ml of DMF, followed by 20 mol% HMAAm and0.01 mol% AIBN. In total, the monomers possessed a molarconcentration of 50 mmol. For the copolymerization, 62°C wasmaintained for 20 h before four freeze-thaw cycles degassed itcompletely. After polymerization, AIBN, unreacted monomers,impurities, and solvent were removed by dialysis against ethanol(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)and distilled water for 7 days. It takes 3 days for the dialyzedsolutions to be lyophilized after they have been dialyzed. Thechemical structure of the copolymer was verified by NMR (JEOL,Tokyo, Japan). The average molecular weight (Mn) andpolydispersity index (PDI) of the copolymer was determinedby gel permeation chromatography (GPC, JASCO International,Tokyo, Japan) using DMF with lithium bromide (LiBr, 10 mm)(Tosho Corporation, Tokyo, Japan) as an eluent sample. A UV-Visible spectrophotometer (JASCO Corporation, Tokyo, Japan)with a heating rate of 1.0 C/min was used to measuretemperature-dependent changes in the transmittance of thecopolymer in phosphate-buffered saline (PBS) (pH = 7.4, 0.1%w/v). We defined the lower critical solution temperature of thecopolymer as the temperature at which 50% of the transmissionwas achieved.Fabrication of fiber meshesA solution for electrospinning was prepared by dissolvingpoly (NIPAAm-co-HMAAm) in HFIP (20 w/v%). Theelectrospun solution contained 30 w/w%, 1.0 w/w%, 0–20 w/w% of MNPs, PTX, and polyvinyl alcohol, respectively. Thesolution was electrospun into fibers using an applied voltageof 20 kV with a flow rate set to 1.0 ml/h, and 13 cm separation ofthe needle (25 gauge) and collector plate at 25°C and 42%humidity (Nanon-01A, MECC Co., Ltd, Fukuoka, Japan). Inorder to remove organic solvents from electrospun fibers, theywere placed in an oven (Tokyo Rikakikai Co., Ltd, Tokyo, Japan)at 140°C for 24 h. This process involved the methylal group inHMAAm and the hydroxy group in PVA were crosslinked in thenanofiber mesh. The morphology of fibers was observed using ascanning electron microscope (SEM) (SU8000, Hitachi High-FIGURE 5Cell viability of SKOV3 cells treated with (A) different concentrations of PTX, and (B) nanofiber meshes with different cycles of AFM. (Data aremean ± SD n = 6, **p < 0.01).Frontiers in Bioengineering and Biotechnology frontiersin.org08Chen et al. 10.3389/fbioe.2022.1046147https://www.frontiersin.org/journals/bioengineering-and-biotechnologyhttps://www.frontiersin.orghttps://doi.org/10.3389/fbioe.2022.1046147Technologies Corporation, Tokyo, Japan) with secondaryelectrons (SE) after Pt coating of the fiber surface. Thediameter of fibers was calculated from a SEM image usingImageJ software. Crosslinking between methylal groups ofHMAAm within the fiber was confirmed based on thedisappearance of absorbance at 1,050 cm−1 through ATR-FTIRspectroscopy (IRPrestige-21, Shimadzu, Kyoto, Japan). Analysiswas conducted after the residual solvent was removed from fibersby vacuum drying (ULVAC KIKO, Inc, Miyazaki, Japan).Rheological characterization of smartnanofiber meshThe rheological properties were tested using a rheometer(MCR301, Anton Paar Japan K.K, Tokyo, Japan). Strainamplitude-dependent dynamic viscoelasticity, shear-rate-dependent viscosity, and continuous-step strain measurementwere performed. In each measurement, the prepared SNM wasswollen with distilled water and then cut into 1 cm diameter circlesfor measurement. In the strain amplitude-dependent dynamicviscoelasticity measurement, the angular frequency was fixed at10 rad/s, the shear strain was varied logarithmically from 0.01% to2000%, and the storage modulus G’ (Pa) and loss modulus G” (Pa)were measured. The number of measurement points was 32, andthe total measurement time was set to 96 s. In the shear-rate-dependent viscosity measurement, shear stress (Pa) was measuredby varying the shear rate logarithmically from 0.001 s−1 to 100 s−1.The number ofmeasuring points was 26 and the total measurementtime was set to 143 s. In the continuous-step strain measurement,the angular frequency was fixed at 10 rad/s. The shear strain wasrepeated for 120 s at 1% and for 60 s at 1,000% for a total of fourcycles, and the storage modulus G’ (Pa) and loss modulus G” (Pa)were measured. The total number of measurement points was 72,and the total measurement time was 720 s.Nanofiber smart mesh heating profilesMNPs within the nanofiber mesh were studied by applyingAMF to determine their heat-generating properties. SNMs (6.0,9.0, 12.0 mg) of MNPs were placed in the center of HOTSHOT2(Alonics Co., Ltd, Tokyo, Japan) of a customized copper coil thatgenerated AMF (480 A, 281 kHz frequency, 362 W). Wemeasured the heating profiles at 30 and 60 s using an FL-IRthermo-camera (CPA-E6, FLIR Systems Japan K.K, Tokyo,Japan).Drug release from smart nanofiber meshIn 1.0 ml of phosphate-buffered saline (PBS) at roomtemperature, samples of SNM (30 mg) thermo crosslinkedwith 5% PVA were first swollen for 5 min by shaking (at100 rpm) (Lin et al., 2019). As soon as the sample reachedequilibrium, it was placed in the center of a copper coil.Afterward, 0.25 ml of the supernatant was collected, and freshPBS was added to the supernatant that had been irradiated withAMF or cooled for 5 min at room temperature.PTX release profiles were quantified by UV-visiblespectroscopy during each switching cycle. (V-650 spectrophotometer, Jasco, Tokyo. Japan). Eight cycleswere repeated. A cumulative release of PTX was calculatedusing the following equation: Wn � Wn−1 + {Cn − Cn−1(1 − l)}, where Wn (μg) and Cn (μg ml-1) are the cumulative releaseamount and concentration of PTX at the n th (n = 1–16)collection process, respectively, and l is the volume collectedPBS (=0.25 ml).Cell preparationThe cells used in this study were SKOV3 (human ovarianadenocarcinoma). McCoy’s 5 A medium was used as a growthmedium with 10% fetal bovine serum and 1% penicillin-streptomycin. To prepare SKOV3 cells, subcultures in a tissueculture dish (100 mm) were incubated for 2 days at 37°C in 5%CO2 (MCO-170AICUVH, PHC Holdings, Tokyo, Japan). Thefollowing experiments collected the cells from a confluentmonolayer in the dish with 0.25% (w/v) trypsin andresuspended them in 10 ml of growth medium.Drug sensitivity of SKOV3 cellsApproximately 1.0 × 105 SKOV3 cells were seeded on a 96-well plate, 200 µL of the growth medium was added andincubated for 24 h. The medium was exchanged for 96-wellplates which contained 0, 0.001, 0.01, 0.1, 1.0, and 10 μg ml−1of PTX, and incubated for 24 h. After adding Alamar Blue (tenpercent of the medium), each well was incubated at 37°C for4 hours. The number of cells was calculated by measuring thefluorescent intensity at 570 nm excitation and 600 nm emission(Tecan Japan). Cell viability and IC50 were calculated based onconsidering the number of cells in the control group (withoutPTX treatment) as 100% viability.AMF-responsive anti-tumor activity InvitroFor the anticancer experiment of the SNM, SKOV3 cells wereseeded in a 6 mm plate at 2 × 106 cells per well for 24 h.Thereafter, the cells incubated a piece of nanofiber meshcross-linked with 5% of PVA (which had a 30 mg weight,30% MNPs and 1% PTX), which was exposed to AMFFrontiers in Bioengineering and Biotechnology frontiersin.org09Chen et al. 10.3389/fbioe.2022.1046147https://www.frontiersin.org/journals/bioengineering-and-biotechnologyhttps://www.frontiersin.orghttps://doi.org/10.3389/fbioe.2022.1046147irradiation (480 A, amplitude 281 kHz frequency) for 5 min.After further incubation at 37°C for another 24 h, the AlamarBlue assay reagent (10% against medium) was added to each welland incubated for 4 hours at 37°C, according to protocol. The cellnumber was computed from the fluorescent intensity measuredby a fluorescence plate drive set at 570 nm excitation and 600 nmemission.Statistical analysisAll experiments were conducted three times and the dataare presented as means ± standard deviation (SD). Statisticalanalysis was performed using the student t-test and varianceone-way analysis (ANOVA) by Origin version 9.0(Northampton, United States). The difference between theresults was considered to be statistically significant for p <0.01 (**).ConclusionIn the current research, switchable drug release injectablenanofiber platforms were engineered for simultaneous forchemotherapeutic and hyperthermia to cancerous cells. TheSNM were fabricated by an electrospinning method with atemperature-responsive polymer, MNPs, and PTX, and thefiber was further cross-linked with PVA. Nanofibersresponded to alternating “on”, and “off” switches of AMFapplication and the controllable release of the drug from thefibers was observed as a result. Aiming for amplification ofchemotherapeutic potency, MNPs-loaded SNM schemed. TheSNM exposure to AMF enables switchable drug release andgenerates hyperthermia effects, which causes heat-induced cellkilling as well as enhanced PTX chemotherapeutic efficiency.Subsequent investigations approved the validity of our strategichyperthermia in amplifying chemotherapeutic potency; namely,the SNM efficiently induced apoptosis of SKOV3 cells throughthe synergistic anticancer effect arising from hyperthermia andPTX. Hence, the current research not only supported anintelligent nanofiber platform for injectable and controllabledrug release to be fabricated, but also urged the promisingpotential of exploiting combination strategy in advancingtumor therapy.Data availability statementThe original contributions presented in the study areincluded in the article/Supplementary Material, furtherinquiries can be directed to the corresponding author.Author contributionsEexperimental preparation: MT and LC; data analysis: LCand MT; chart production: LC and MT; writing-original draftpreparation: LC andMT; writing-review and editing: LC andME.All authors have read and agreed to the published version of themanuscript.FundingThis study was support by the JSPS KAKENHI Grant-in-Aidfor Scientific Research(B) (JP19H04476) and Grant-in-Aid forTransformative Research Areas(A) (JP20H05877).Conflict of interestThe authors declare that the research was conducted in theabsence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.Publisher’s noteAll claims expressed in this article are solely those of theauthors and do not necessarily represent those of their affiliatedorganizations, or those of the publisher, the editors and thereviewers. 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