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Nobuyoshi Fukumitsu, Yoshitaka Matsumoto, Lili Chen, Yu Sugawara, [Nanami Fujisawa](https://orcid.org/0000-0002-8894-1790), Eri Niiyama, Sosuke Ouchi, Emiho Oe, Takashi Saito, [Mitsuhiro Ebara](https://orcid.org/0000-0002-7906-0350)

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[Development of Layer-by-Layer Magnetic Nanoparticles for Application to Radiotherapy of Pancreatic Cancer](https://mdr.nims.go.jp/datasets/f09032d9-b87f-4708-9aa4-49ca4892e620)

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Development of Layer-by-Layer Magnetic Nanoparticles for Application to Radiotherapy of Pancreatic CancerAcademic Editors: Alexander Kotlyarand Vasco D. B. BonifácioReceived: 27 December 2024Revised: 7 February 2025Accepted: 18 March 2025Published: 20 March 2025Citation: Fukumitsu, N.;Matsumoto, Y.; Chen, L.; Sugawara, Y.;Fujisawa, N.; Niiyama, E.; Ouchi, S.;Oe, E.; Saito, T.; Ebara, M.Development of Layer-by-LayerMagnetic Nanoparticles forApplication to Radiotherapy ofPancreatic Cancer. Molecules 2025, 30,1382. https://doi.org/10.3390/molecules30061382Copyright: © 2025 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license(https://creativecommons.org/licenses/by/4.0/).ArticleDevelopment of Layer-by-Layer Magnetic Nanoparticles forApplication to Radiotherapy of Pancreatic CancerNobuyoshi Fukumitsu 1,† , Yoshitaka Matsumoto 2,3,*,† , Lili Chen 4 , Yu Sugawara 3, Nanami Fujisawa 4 ,Eri Niiyama 4, Sosuke Ouchi 4, Emiho Oe 4 , Takashi Saito 2,3 and Mitsuhiro Ebara 41 Department of Radiation Oncology, Kobe Proton Center, Kobe 650-0047, Japan; fukumitsun@yahoo.co.jp2 Department of Radiation Oncology, Clinical Medicine, Faculty of Medicine, University of Tsukuba,Tsukuba 305-8575, Japan; saitoh@pmrc.tsukuba.ac.jp3 Proton Medical Research Center, University of Tsukuba Hospital, Tsukuba 305-8576, Japan;yu-suga0315@outlook.com4 Smart Polymers Group, Research Center for Macromolecules and Biomaterials, National Institute forMaterials Science, Tsukuba 305-0044, Japan; chenlili03070526@hotmail.com (L.C.);nfujisawa.tokyo@gmail.com (N.F.); lab.sosuke@gmail.com (S.O.); emiho0219@gmail.com (E.O.);ebara.mitsuhiro@nims.go.jp (M.E.)* Correspondence: ymatsumoto@pmrc.tsukuba.ac.jp; Tel.: +81-29-853-7100† These authors contributed equally to this work.Abstract: Pancreatic cancer is among the deadliest malignancies, with few treatmentoptions for locally advanced, unresectable cases. Conventional therapies, such as chemora-diotherapy and hyperthermia, show promise but face challenges in improving outcomes.This study introduces a novel drug delivery system using gemcitabine (GEM)-loadedlayer-by-layer magnetic nanoparticles (LBL MNPs) combined with alternating magneticfield (AMF) application and X-ray irradiation to enhance therapeutic efficacy. LBL MNPswere synthesized using optimized layering techniques to achieve superior drug loadingand controlled release. Human pancreatic cancer cells (PANC-1) were treated with LBLMNPs alone, with AMF-induced hyperthermia, and in combination with X-rays. Theresults demonstrate that the 7-layer LBL MNPs exhibited optimal cytotoxicity, significantlyreducing cell viability at concentrations of 30 µg/mL and higher. Combining 7-layer LBLMNPs with AMF increased cell death in a time- and concentration-dependent manner,achieving up to 98% inhibition of cell proliferation. The addition of X-rays to the regimendemonstrated a strong synergistic effect, resulting in a 13-fold increase in cell death com-pared to controls. These findings highlight the potential of this integrated approach toimprove outcomes in patients with pancreatic cancer.Keywords: layer-by-layer magnetic nanoparticles; radiosensitization; thermo-chemotherapy1. IntroductionPancreatic cancer has a poor prognosis, with the annual death toll steadily rising toapproximately 38,000 in 2021, making it the third leading cause of cancer-related deaths inJapan, according to Cancer Statistics in Japan [1]. Surgical resection is currently the onlycurative treatment; however, less than 20% of cases are resectable at presentation. Addi-tionally, up to 25–30% of newly diagnosed patients with non-distant metastatic lesions areclassified as borderline resectable or unresectable based on the extent of vascular involve-ment. In these unresectable cases, chemotherapy or chemoradiotherapy is administered [2].Despite advancements in drug development and radiotherapy techniques over recentMolecules 2025, 30, 1382 https://doi.org/10.3390/molecules30061382https://doi.org/10.3390/molecules30061382https://doi.org/10.3390/molecules30061382https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://www.mdpi.com/journal/moleculeshttps://www.mdpi.comhttps://orcid.org/0000-0003-2417-8199https://orcid.org/0000-0002-2074-3042https://orcid.org/0000-0001-8290-5230https://orcid.org/0000-0002-8894-1790https://orcid.org/0000-0001-7326-7740https://orcid.org/0009-0006-4663-5580https://orcid.org/0000-0002-7906-0350https://doi.org/10.3390/molecules30061382https://www.mdpi.com/article/10.3390/molecules30061382?type=check_update&version=1Molecules 2025, 30, 1382 2 of 13decades, the prognosis for pancreatic cancer remains poor. Therefore, the development ofalternative treatments combining chemotherapy and radiotherapy is urgently needed [3].Hyperthermia has a sensitizing effect on chemotherapy and radiotherapy, potentiallyimproving the efficacy of these therapies for diseases that are highly resistant to treatment.Recent studies have demonstrated that subablative hyperthermia at approximately 42 ◦Csignificantly enhances chemotherapeutic effects by increasing the sensitivity of cancercells to chemotherapeutic drugs and reversing chemoresistance [4–9]. Although biologicalevidence suggests that hyperthermia is effective for the treatment of cancer, its widespreadclinical acceptance remains limited. This is primarily due to the challenge of selectively heat-ing the cancer lesion while minimizing effects on surrounding normal tissues, particularlyfor deep-seated tumors such as pancreatic cancer.In this study, hyperthermia and chemotherapy, along with various materials anddrug-delivery techniques, were developed [10–17]. The layer-by-layer (LBL) method,innovatively introduced by Decher et al., has been extended due to its operational simplic-ity [18,19]. To achieve precise drug delivery, functional material units must be designed foreach component, and carriers that allow flexible control over the structure are highly bene-ficial. In this study, magnetic nanoparticles were selected as the heat source, and inductionheating through irradiation with an alternating magnetic field was utilized. Furthermore,for use in combination with chemotherapy, a polyelectrolyte was coated onto the surface ofthe magnetic nanoparticles via electrostatic interactions, with the nanoparticles serving asthe core and the outermost shell covered with gemcitabine (GEM), a drug delivery carrierused in the treatment of pancreatic cancer (Figure 1).Molecules 2025, 30, x FOR PEER REVIEW 2 of 14   techniques over recent decades, the prognosis for pancreatic cancer remains poor. Therefore, the development of alternative treatments combining chemotherapy and radiotherapy is urgently needed [3]. Hyperthermia has a sensitizing effect on chemotherapy and radiotherapy, potentially improving the efficacy of these therapies for diseases that are highly resistant to treatment. Recent studies have demonstrated that subablative hyperthermia at approximately 42 °C significantly enhances chemotherapeutic effects by increasing the sensitivity of cancer cells to chemotherapeutic drugs and reversing chemoresistance [4–9]. Although biological evidence suggests that hyperthermia is effective for the treatment of cancer, its widespread clinical acceptance remains limited. This is primarily due to the challenge of selectively heating the cancer lesion while minimizing effects on surrounding normal tissues, particularly for deep-seated tumors such as pancreatic cancer. In this study, hyperthermia and chemotherapy, along with various materials and drug-delivery techniques, were developed [10–17]. The layer-by-layer (LBL) method, innovatively introduced by Decher et al., has been extended due to its operational simplicity [18,19]. To achieve precise drug delivery, functional material units must be designed for each component, and carriers that allow flexible control over the structure are highly beneficial. In this study, magnetic nanoparticles were selected as the heat source, and induction heating through irradiation with an alternating magnetic field was utilized. Furthermore, for use in combination with chemotherapy, a polyelectrolyte was coated onto the surface of the magnetic nanoparticles via electrostatic interactions, with the nanoparticles serving as the core and the outermost shell covered with gemcitabine (GEM), a drug delivery carrier used in the treatment of pancreatic cancer (Figure 1).  Figure 1. Schematic of layer-by-layer nanoparticle preparation combined with thermal chemoradiotherapy for efficient synergistic treatment of pancreatic cancer. 2. Results and Discussion 2.1. Characterization of the Particle Size and Potential of LBL MNPs The physical and chemical properties of nanocarriers are crucial for their cellular internalization. For example, magnetic nanoparticle (MNP) sizes between 50 and 200 nm are more likely to accumulate at tumor sites through the enhanced permeability and retention (EPR) effect and be internalized by tumor cells [20]. Dynamic light scattering Figure 1. Schematic of layer-by-layer nanoparticle preparation combined with thermal chemoradio-therapy for efficient synergistic treatment of pancreatic cancer.2. Results and Discussion2.1. Characterization of the Particle Size and Potential of LBL MNPsThe physical and chemical properties of nanocarriers are crucial for their cellularinternalization. For example, magnetic nanoparticle (MNP) sizes between 50 and 200 nmare more likely to accumulate at tumor sites through the enhanced permeability andretention (EPR) effect and be internalized by tumor cells [20]. Dynamic light scattering(DLS) was employed to determine the particle size, polydispersity index (PDI), and zetapotential (surface charge) of the LBL MNPs with varying numbers of layers.Molecules 2025, 30, 1382 3 of 13As shown in Figure 2, the particle sizes of LBL MNPs with different numbers oflayers were measured to reflect electrostatic interactions between the positively chargedpolcation and negatively charged polyanion. This change in particle size is likely due tothe introduction of the negatively charged polymer, poly4-styrenesulfonate (PSS), and thepositively charged polyallylamine (PAH). Simultaneously, the particle size of the MNPsincreased as each polymer layer was added, confirming the successful preparation of LBLMNPs (Figure 2a). Specifically, the particle sizes of the polymers in the 5-, 7-, and 9-layerconfigurations were 157.6 ± 65.7 nm, 198.7 ± 24.2 nm, and 369.0 ± 199.1 nm, respectively,and the PDIs were 1.2 ± 1.6, 0.8 ± 0.5, and 1.22 ± 1.2, respectively. Additionally, thezeta potentials for the 5-, 7-, and 9-layer polymers were −38.0 ± 17.4 mV, −15.6 ± 5.5 mV,and −50.7 ± 13.9 mV, respectively, all of which were negative, facilitating subsequentelectrostatic loading of the positively charged GEM (Figure 2b). Transmission electronmicroscopy (TEM) images of the 1- and 7-layer coated LBL MNPs were also acquired tovisualize the polymer layers (Figure 2c,d).Molecules 2025, 30, x FOR PEER REVIEW 3 of 14   (DLS) was employed to determine the particle size, polydispersity index (PDI), and zeta potential (surface charge) of the LBL MNPs with varying numbers of layers. As shown in Figure 2, the particle sizes of LBL MNPs with different numbers of layers were measured to reflect electrostatic interactions between the positively charged polcation and negatively charged polyanion. This change in particle size is likely due to the introduction of the negatively charged polymer, poly4-styrenesulfonate (PSS), and the positively charged polyallylamine (PAH). Simultaneously, the particle size of the MNPs increased as each polymer layer was added, confirming the successful preparation of LBL MNPs (Figure 2a). Specifically, the particle sizes of the polymers in the 5-, 7-, and 9-layer configurations were 157.6 ± 65.7 nm, 198.7 ± 24.2 nm, and 369.0 ± 199.1 nm, respectively, and the PDIs were 1.2 ± 1.6, 0.8 ± 0.5, and 1.22 ± 1.2, respectively. Additionally, the zeta potentials for the 5-, 7-, and 9-layer polymers were −38.0 ± 17.4 mV, −15.6 ± 5.5 mV, and -50.7 ± 13.9 mV, respectively, all of which were negative, facilitating subsequent electrostatic loading of the positively charged GEM (Figure 2b). Transmission electron microscopy (TEM) images of the 1- and 7-layer coated LBL MNPs were also acquired to visualize the polymer layers (Figure 2c,d).   (a) (b)   (c) (d) Figure 2. Changes in particle size (a) and zeta potential (b) for LBL MNPs. TEM images of LBL MNPs with 1- (c) and 7- (d) layers. 2.2. Heat Generation Behavior of LBL MNPs Magnetic hyperthermia, which utilizes MNPs as a heat source, is a technique that transfers heat to tumor cells. This method generates heat through the interaction with alternating magnetic fields (AMF), making it an effective treatment for targeting deep-Figure 2. Changes in particle size (a) and zeta potential (b) for LBL MNPs. TEM images of LBL MNPswith 1- (c) and 7- (d) layers.2.2. Heat Generation Behavior of LBL MNPsMagnetic hyperthermia, which utilizes MNPs as a heat source, is a technique thattransfers heat to tumor cells. This method generates heat through the interaction withalternating magnetic fields (AMF), making it an effective treatment for targeting deep-seated, inaccessible tumors. Heat generation occurs when magnetic nanoparticles areexposed to a magnetic field, with energy being converted to heat during relaxation processesMolecules 2025, 30, 1382 4 of 13based on the Néel and Brownian relaxation mechanisms. Therefore, it is essential to useAMF rather than direct-current magnetic fields to ensure sustained heat generation. Theheat-generation behavior of polymer-coated LBL MNPs under AMF exposure was observed,as shown in Figure 3. When the MNPs were subjected to AMF for 240 s, the temperatureof the LBL MNPs suspension increased from 29.0 to 42.3 ◦C. This temperature increaseis similar to that of bare MNPs. Hyperthermia requires a temperature range of 42–45 ◦C,and the heat generated by LBL MNPs falls within this range, making them suitable forhyperthermia applications. Eddy currents induced by magnetic fields can affect tissues andorgans, potentially causing carbonization and necrosis. However, the frequency used inthis study was only 281 kHz, which is well below the safe frequency threshold for AMF(100–300 kHz) [21].Molecules 2025, 30, x FOR PEER REVIEW 4 of 14   seated, inaccessible tumors. Heat generation occurs when magnetic nanoparticles are exposed to a magnetic field, with energy being converted to heat during relaxation processes based on the Néel and Brownian relaxation mechanisms. Therefore, it is essential to use AMF rather than direct-current magnetic fields to ensure sustained heat generation. The heat-generation behavior of polymer-coated LBL MNPs under AMF exposure was observed, as shown in Figure 3. When the MNPs were subjected to AMF for 240 s, the temperature of the LBL MNPs suspension increased from 29.0 to 42.3 °C. This temperature increase is similar to that of bare MNPs. Hyperthermia requires a temperature range of 42–45 °C, and the heat generated by LBL MNPs falls within this range, making them suitable for hyperthermia applications. Eddy currents induced by magnetic fields can affect tissues and organs, potentially causing carbonization and necrosis. However, the frequency used in this study was only 281 kHz, which is well below the safe frequency threshold for AMF (100–300 kHz) [21].   (a) (b)  (c) Figure 3. Heat generation behavior of LBL MNPs coated with polymers when exposed to an AMF field. Heatmap images were obtained when particles were exposed for (a) 0 and (b) 4 min. MNPs coated with up to 7 layers of polymer MNP suspension in water are shown. (c) Data showing the temperature increase per min for LBL MNPs (●) and MNPs (◾). 2.3. Relationship Between Cell Killing Effect of LBL MNPs, Concentration, and Layer Number The drug release curves of GEM encapsulated in LBL MNPs are shown in Figure 4a. GEM loading increased with the number of layers, with a release of 7.61 µg/mL GEM after 24 h in a 100 µg/mL suspension of 7-layer particles (Figure 4a). When PANC-1 cells were treated with LBL MNPs of different layer numbers, a concentration-dependent increase Figure 3. Heat generation behavior of LBL MNPs coated with polymers when exposed to an AMFfield. Heatmap images were obtained when particles were exposed for (a) 0 and (b) 4 min. MNPscoated with up to 7 layers of polymer MNP suspension in water are shown. (c) Data showing thetemperature increase per min for LBL MNPs (•) and MNPs (■).2.3. Relationship Between Cell Killing Effect of LBL MNPs, Concentration, and Layer NumberThe drug release curves of GEM encapsulated in LBL MNPs are shown in Figure 4a.GEM loading increased with the number of layers, with a release of 7.61 µg/mL GEM after24 h in a 100 µg/mL suspension of 7-layer particles (Figure 4a). When PANC-1 cells weretreated with LBL MNPs of different layer numbers, a concentration-dependent increase incytotoxicity was observed for each layer (Figure 4b). Regarding the drug release behavior,GEM on the LBL particles was electrostatically bound; therefore, it is likely that it was grad-Molecules 2025, 30, 1382 5 of 13ually released by displacement with other electrolytes. Results from the two-way analysisof variance (ANOVA) followed by a Bonferroni post-hoc test indicate that both the numberof layers (F (3, 48) = 13.349, p < 0.001, η2 = 0.455) and concentration (F (5, 48) = 151.989,p < 0.001, η2 = 0.941) significantly affected survival. Additionally, a significant interactioneffect between layer number and concentration was observed (F (15, 48) = 2.881, p = 0.003,η2 = 0.474), suggesting that the concentration effect on survival varies depending on thelayer type. Post-hoc Bonferroni tests revealed significant differences in survival acrossdifferent concentration levels (p < 0.001). Higher concentrations resulted in significantlylower survival rates, with a steep decline from 0 to 100 µg/mL. Among the different lay-ers, 7 layers showed the most distinct behavior, exhibiting significantly lower survivalrates compared to the 5, 9, and 11 layers (p < 0.001). The differences between the 9 and11 layers were not statistically significant (Figure 4b). The superior performance of 7-layerLBL MNPs may be attributed to an optimal balance between drug-loading capacity andcontrolled release. In addition, the significant interaction effect (p = 0.003) suggests thatthe impact of concentration on survival varies depending on the layer structure. Notably,7 layers exhibited the most pronounced decrease in survival, indicating that the specificstructural or compositional properties of this layer may enhance drug penetration or toxic-ity. Studies have shown that the number of layers in multilayered nanoparticles directlyaffects drug release profiles and cellular uptake efficiency [22,23]. A 7-layer configurationlikely provides sufficient drug-loading sites while maintaining structural integrity, whichfacilitates efficient cellular internalization. In contrast, both under-structured (e.g., the5-layer) and over-structured (e.g., 9- and 11-layer) MNPs may exhibit suboptimal perfor-mance. In particular, the zeta potential of the 5- and 9-layer MNPs showed a relativelystrong negative surface charge, suggesting difficulty in GEM release. On the other hand,over-structured polymers may hinder drug release due to excessive layering. These stud-ies demonstrate that the number of nanoparticle layers significantly affects drug releaseprofiles and cellular uptake efficiency, supporting the idea that 7-layer LBL nanoparticlesstrike an optimal balance.Molecules 2025, 30, x FOR PEER REVIEW 5 of 14   in cytotoxicity was observed for each layer (Figure 4b). Regarding the drug release behavior, GEM on the LBL particles was electrostatically bound; therefore, it is likely that it was gradually released by displacement with other electrolytes. Results from the two-way analysis of variance (ANOVA) followed by a Bonferroni post-hoc test indicate that both the number of layers (F (3, 48) = 13.349, p < 0.001, η2 = 0.455) and concentration (F (5, 48) = 151.989, p < 0.001, η2 = 0.941) significantly affected survival. Additionally, a significant interaction effect between layer number and concentration was observed (F (15, 48) = 2.881, p = 0.003, η2 = 0.474), suggesting that the concentration effect on survival varies depending on the layer type. Post-hoc Bonferroni tests revealed significant differences in survival across different concentration levels (p < 0.001). Higher concentrations resulted in significantly lower survival rates, with a steep decline from 0 to 100 µg/mL. Among the different layers, 7 layers showed the most distinct behavior, exhibiting significantly lower survival rates compared to the 5, 9, and 11 layers (p < 0.001). The differences between the 9 and 11 layers were not statistically significant (Figure 4b). The superior performance of 7-layer LBL MNPs may be attributed to an optimal balance between drug-loading capacity and controlled release. In addition, the significant interaction effect (p = 0.003) suggests that the impact of concentration on survival varies depending on the layer structure. Notably, 7 layers exhibited the most pronounced decrease in survival, indicating that the specific structural or compositional properties of this layer may enhance drug penetration or toxicity. Studies have shown that the number of layers in multilayered nanoparticles directly affects drug release profiles and cellular uptake efficiency [22,23]. A 7-layer configuration likely provides sufficient drug-loading sites while maintaining structural integrity, which facilitates efficient cellular internalization. In contrast, both under-structured (e.g., the 5-layer) and over-structured (e.g., 9- and 11-layer) MNPs may exhibit suboptimal performance. In particular, the zeta potential of the 5- and 9-layer MNPs showed a relatively strong negative surface charge, suggesting difficulty in GEM release. On the other hand, over-structured polymers may hinder drug release due to excessive layering. These studies demonstrate that the number of nanoparticle layers significantly affects drug release profiles and cellular uptake efficiency, supporting the idea that 7-layer LBL nanoparticles strike an optimal balance.   (a) (b) Figure 4. (a) Drug release profile of layer-by-layer magnetic nanoparticles (LBL MNPs) and (b) the effect of LBL MNPs on cell viability. The concentration of released GEM (µg/mL) was measured over time (h) for nanoparticles with different numbers of layers (1, 3, 5, and 7). GEM release showed a layer-dependent profile, with nanoparticles having more layers exhibiting a more sustained release. The data indicate that increasing the number of layers results in prolonged drug release. Cell viability (relative luminescence units, RLU) was assessed after treatment with LBL MNPs at various concentrations (0, 1, 3, 10, 30, and 100 µg/mL). Different numbers of layers (5-, 7-, 9-, and 11-layer) were evaluated for their cytotoxic effects. Data are presented as the mean ± standard error Figure 4. (a) Drug release profile of layer-by-layer magnetic nanoparticles (LBL MNPs) and (b) theeffect of LBL MNPs on cell viability. The concentration of released GEM (µg/mL) was measured overtime (h) for nanoparticles with different numbers of layers (1, 3, 5, and 7). GEM release showed alayer-dependent profile, with nanoparticles having more layers exhibiting a more sustained release.The data indicate that increasing the number of layers results in prolonged drug release. Cellviability (relative luminescence units, RLU) was assessed after treatment with LBL MNPs at variousconcentrations (0, 1, 3, 10, 30, and 100 µg/mL). Different numbers of layers (5-, 7-, 9-, and 11-layer)were evaluated for their cytotoxic effects. Data are presented as the mean ± standard error (SE)from three independent experiments (n = 3). Cell viability decreased with increasing LBL MNPconcentration, with variations observed depending on the number of layers.Molecules 2025, 30, 1382 6 of 132.4. Enhancement of the Synergistic Cell-Killing Effect by LBL MNPs and AMF ApplicationCombining 7-layer LBL MNPs with magnetic hyperthermia confirmed that the inhibi-tion of cell proliferation activity was dependent on both the LBL MNPs concentration andAMF application time. Combining 100 µg/mL LBL MNPs with 30 min of AMF treatmentresulted in approximately 98% inhibition of cell proliferation compared to the absence ofLBL MNPs. Similarly, the combined use of LBL MNPs and AMF significantly increased thenumber of test cells in a concentration- and time-dependent manner. A two-way ANOVA re-vealed significant main effects of LBL MNP concentration (η2 = 0.997 for viability, η2 = 0.952for cell death) and AMF application time (η2 = 0.975 for viability, η2 = 0.913 for cell death)on cell viability and cell death induction. A significant interaction between these factorswas observed for both viability (η2 = 0.923, p < 0.001) and cell death (η2 = 0.811, p < 0.001),indicating that AMF enhances the cytotoxic effects of LBL MNPs. Post hoc analysis revealeda dose-dependent decrease in viability, with significant reductions observed starting from10.00 µg/mL LBL MNPs and 5.00 min of AMF application. However, a plateau effect wasnoted at 30.00 µg/mL and above for LBL MNPs and at longer AMF application times,suggesting a saturation of cytotoxic effects. Similarly, cell death significantly increasedwith LBL MNP concentration and AMF application, showing a synergistic effect betweenthese two factors (Figure 5). These findings suggest that AMF exposure enhances LBLMNPs-induced cytotoxicity, likely through increased intracellular nanoparticle uptake andhyperthermia effects [24]. The observed saturation effect is consistent with previous studiesshowing that beyond a threshold, additional drug exposure does not significantly enhanceapoptosis [25]. Similarly, the plateau effect at higher AMF intensities aligns with reportsthat hyperthermia-induced cytotoxicity has an upper limit [26]. The synergistic interactionsupports previous findings that magnetic hyperthermia enhances chemotherapy by increas-ing reactive oxygen species (ROS) generation and mitochondrial damage [27]. LBL MNPsinduce a local temperature rise through AMF application, inducing apoptosis and necrosisof tumor cells [28,29]. In this study, cell proliferation inhibition and cell death inductionwere enhanced with increasing LBL MNPs concentration and AMF irradiation time, whichis thought to be related to the heating effect of LBL MNPs by AMF. In particular, theinhibition of cell proliferation reached approximately 98% after combining 100 µg/mL LBLMNPs with 30 min of AMF application, indicating that the combination of an appropriateLBL MNPs concentration and AMF application time may maximize the therapeutic effect.Hyperthermia induced by MNPs occurs because temperature increases the permeability oftumor cells and their sensitivity to chemotherapy [30]. These results suggest that treatmentwith LBL MNPs exposed to AMF made cancer cells more sensitive to chemotherapy andthus efficiently induced cell death [30].2.5. Enhancement of the Synergistic Cell-Killing Effect by LBL MNPs, AMF, andX-Ray IrradiationA combination of radiation therapy and chemotherapy can enhance the therapeuticeffect and is widely used in clinical practice. The synergistic effect of these therapiesis expected to result in better proliferation inhibition of cancer cells and more efficienttreatment. GEM, in addition to its cytotoxic effects, is also a potent radiosensitizer. Suchnucleotide derivatives are attractive when used in combination with radiotherapy. Dueto their preferential cytotoxicity against proliferating cells, these derivatives may alsoreduce cancer cell proliferation and slow cellular repopulation during radiotherapy (RT).However, hypoxic cancer cells are radio-tolerant but sensitive to heat [31]. We confirmedthe enhancement of the cell-killing effect by adding X-ray irradiation to the LBL MNPstreatment and AMF application. As a result, significant inhibition of cell proliferation wasconfirmed compared to the combined use of the two treatments. In particular, in the groupMolecules 2025, 30, 1382 7 of 13where AMF was applied for 30 min, an enhancement effect was confirmed that inhibitedproliferation activity by more than 80% even in the low concentration (5 µg/mL) LBLMNPs group, and an enhancement effect of more than 95% was confirmed in the highconcentration (100 µg/mL) group. Similarly, the induction of cell death was confirmedusing a combination of these three treatments. The combination of LBL MNPs and 5 GyX-rays also showed a maximum cell death induction effect of approximately 3.7 timescompared to the control group. The effect was enhanced depending on the LBL MNPsconcentration and AMF application time, and the combination of 100 µg/mL LBL MNPs,30 min of AMF application, and X-rays induced cell death 13 times higher than that ofthe control (Figure 5b). A three-way ANOVA revealed significant main effects of X-rayirradiation (η2 = 0.967 for viability, η2 = 0.933 for cell death), LBL MNPs concentration(η2 = 0.996 for viability, η2 = 0.933 for cell death), and AMF intensity (η2 = 0.805 for viability,η2 = 0.791 for cell death). Importantly, significant interaction effects were observed forX-ray and drug concentration (η2 = 0.924 for viability, η2 = 0.875 for cell death), X-rayand AMF (η2 = 0.805 for viability, η2 = 0.550 for cell death), and drug concentrationand AMF (η2 = 0.923 for viability, η2 = 0.712 for cell death). A three-way interaction(η2 = 0.881 for viability, η2 = 0.551 for cell death, p < 0.001) was also observed, indicatingthat combining all three factors results in the greatest cytotoxic effect. Post hoc analysesrevealed that X-ray irradiation significantly enhances the effects of both LBL MNPs andAMF (Figure 6). Although only LBL MNPs and AMF reduce viability and increase cell death,the addition of X-rays further amplifies these effects, likely due to increased DNA damageand apoptosis induction. The strong interaction effects suggest that these treatments worksynergistically rather than additively. These results are consistent with prior findingsthat radiotherapy induces DNA double-strand breaks, enhancing chemotherapy-inducedapoptosis [32]. Furthermore, AMF exposure has been shown to enhance nanoparticle-mediated hyperthermia, which can increase ROS generation and further sensitize cancercells to radiation [33]. The significant three-way interaction suggests that LBL MNPs, AMF,and X-ray irradiation together create a potent combination for maximizing cancer celldeath [34]. It has been reported that hyperthermia using LBL MNPs and AMF enhancesthe therapeutic effect on tumor cells when combined with radiotherapy [35]. DNA double-strand breaks are a major cause of radiation-induced cell death. Nucleotide derivativessuch as GEM, as DNA synthesis inhibitors, have the potential to inhibit the repair ofradiation-induced DNA damage. Once incorporated into the DNA repair patch, nucleosideanalogs may trigger an apoptotic response similar to that observed during replication.DNA damage is induced by radiation at all stages of the cell cycle, where the cell cycleenters the S phase arrest mode, and cells in the S phase are most sensitive to heat [36].This mechanism suggests the possibility of extending the cytotoxicity of these analogsto cells outside the S phase [31]. In this study, cell proliferation inhibition and cell deathinduction were significantly enhanced by combining LBL MNPs and AMF with X-rayirradiation. In particular, even at a low concentration (5 µg/mL) of LBL MNPs, theinhibition of cell proliferation reached 80% or more after 30 min of AMF application andX-ray irradiation, indicating the potential for achieving a high therapeutic effect whilereducing the concentration of LBL MNPs. In addition, the cell death induction effectreached 13 times that of the control group by combining a high concentration (100 µg/mL)of LBL MNPs, 30 min of AMF application, and 5 Gy of X-ray irradiation, suggesting astrong synergistic effect from the combination of all three treatments (Figures 5 and 6).Molecules 2025, 30, 1382 8 of 13Molecules 2025, 30, x FOR PEER REVIEW 7 of 14     (a) (b) Figure 5. Effects of layer-by-layer magnetic nanoparticles (LBL MNPs) on (a) cell viability and (b) cell death induction. (a) Cell viability and (b) cell death induction were assessed after treatment with 7-layer LBL-MNPs at various concentrations (0, 10, 30, and 100 µg/mL) and different alternating magnetic field (AMF) exposure times (0, 5, 10, 20, and 30 min). After 24 h of treatment, cell viability decreased in a concentration- and time-dependent manner, whereas cell death induction increased accordingly. Data are presented as mean ± standard error (SE) from three independent experiments (n = 3). 2.5. Enhancement of the Synergistic Cell-Killing Effect by LBL MNPs, AMF, and X-Ray Irradiation A combination of radiation therapy and chemotherapy can enhance the therapeutic effect and is widely used in clinical practice. The synergistic effect of these therapies is expected to result in better proliferation inhibition of cancer cells and more efficient treatment. GEM, in addition to its cytotoxic effects, is also a potent radiosensitizer. Such nucleotide derivatives are attractive when used in combination with radiotherapy. Due to their preferential cytotoxicity against proliferating cells, these derivatives may also reduce cancer cell proliferation and slow cellular repopulation during radiotherapy (RT). However, hypoxic cancer cells are radio-tolerant but sensitive to heat [31]. We confirmed the enhancement of the cell-killing effect by adding X-ray irradiation to the LBL MNPs treatment and AMF application. As a result, significant inhibition of cell proliferation was confirmed compared to the combined use of the two treatments. In particular, in the group where AMF was applied for 30 min, an enhancement effect was confirmed that inhibited proliferation activity by more than 80% even in the low concentration (5 µg/mL) LBL MNPs group, and an enhancement effect of more than 95% was confirmed in the high concentration (100 µg/mL) group. Similarly, the induction of cell death was confirmed using a combination of these three treatments. The combination of LBL MNPs and 5 Gy X-rays also showed a maximum cell death induction effect of approximately 3.7 times compared to the control group. The effect was enhanced depending on the LBL MNPs concentration and AMF application time, and the combination of 100 µg/mL LBL MNPs, 30 min of AMF application, and X-rays induced cell death 13 times higher than that of the control (Figure 5b). A three-way ANOVA revealed significant main effects of X-ray irradiation (η² = 0.967 for viability, η² = 0.933 for cell death), LBL MNPs concentration (η² = 0.996 for viability, η² = 0.933 for cell death), and AMF intensity (η² = 0.805 for viability, η² = 0.791 for cell death). Importantly, significant interaction effects were observed for X-ray and drug concentration (η² = 0.924 for viability, η² = 0.875 for cell death), X-ray and AMF (η² = 0.805 for viability, η² = 0.550 for cell death), and drug concentration and AMF (η² = 0.923 for viability, η² = 0.712 for cell death). A three-way interaction (η² = 0.881 for viability, η² = 0.551 for cell death, p < 0.001) was also observed, indicating that combining all three factors results in the greatest cytotoxic effect. Post hoc analyses revealed that X-Figure 5. Effects of layer-by-layer magnetic nanoparticles (LBL MNPs) on (a) cell viability and (b) celldeath induction. (a) Cell viability and (b) cell death induction were assessed after treatment with7-layer LBL-MNPs at various concentrations (0, 10, 30, and 100 µg/mL) and different alternatingmagnetic field (AMF) exposure times (0, 5, 10, 20, and 30 min). After 24 h of treatment, cell viabilitydecreased in a concentration- and time-dependent manner, whereas cell death induction increasedaccordingly. Data are presented as mean ± standard error (SE) from three independent experiments(n = 3).Molecules 2025, 30, x FOR PEER REVIEW 8 of 14   ray irradiation significantly enhances the effects of both LBL MNPs and AMF (Figure 6). Although only LBL MNPs and AMF reduce viability and increase cell death, the addition of X-rays further amplifies these effects, likely due to increased DNA damage and apoptosis induction. The strong interaction effects suggest that these treatments work synergistically rather than additively. These results are consistent with prior findings that radiotherapy induces DNA double-strand breaks, enhancing chemotherapy-induced apoptosis [32]. Furthermore, AMF exposure has been shown to enhance nanoparticle-mediated hyperthermia, which can increase ROS generation and further sensitize cancer cells to radiation [33]. The significant three-way interaction suggests that LBL MNPs, AMF, and X-ray irradiation together create a potent combination for maximizing cancer cell death [34]. It has been reported that hyperthermia using LBL MNPs and AMF enhances the therapeutic effect on tumor cells when combined with radiotherapy [35]. DNA double-strand breaks are a major cause of radiation-induced cell death. Nucleotide derivatives such as GEM, as DNA synthesis inhibitors, have the potential to inhibit the repair of radiation-induced DNA damage. Once incorporated into the DNA repair patch, nucleoside analogs may trigger an apoptotic response similar to that observed during replication. DNA damage is induced by radiation at all stages of the cell cycle, where the cell cycle enters the S phase arrest mode, and cells in the S phase are most sensitive to heat [36]. This mechanism suggests the possibility of extending the cytotoxicity of these analogs to cells outside the S phase [31]. In this study, cell proliferation inhibition and cell death induction were significantly enhanced by combining LBL MNPs and AMF with X-ray irradiation. In particular, even at a low concentration (5 µg/mL) of LBL MNPs, the inhibition of cell proliferation reached 80% or more after 30 min of AMF application and X-ray irradiation, indicating the potential for achieving a high therapeutic effect while reducing the concentration of LBL MNPs. In addition, the cell death induction effect reached 13 times that of the control group by combining a high concentration (100 µg/mL) of LBL MNPs, 30 min of AMF application, and 5 Gy of X-ray irradiation, suggesting a strong synergistic effect from the combination of all three treatments (Figures 5 and 6).   (a) (b) Figure 6. Effects of layer-by-layer magnetic nanoparticles (LBL MNPs) on (a) cell viability and (b) cell death induction. (a) Cell viability and (b) cell death induction were assessed after treatment with 7-layer LBL MNPs at various concentrations (0, 10, 30, and 100 µg/mL) and different alternating magnetic field (AMF) exposure times (0, 5, 10, 20, and 30 min) and X-ray irradiation (5 Gy). After 24 h of treatment, (a) cell viability decreased in a concentration- and time-dependent manner, whereas (b) cell death induction increased accordingly. Data are presented as mean ± standard error (SE) from three independent experiments (n = 3). Surgery, chemotherapy, and radiation therapy are the most common cancer treatments currently available, with several new therapies under development [37–39]. Figure 6. Effects of layer-by-layer magnetic nanoparticles (LBL MNPs) on (a) cell viability and (b) celldeath induction. (a) Cell viability and (b) cell death induction were assessed after treatment with7-layer LBL MNPs at various concentrations (0, 10, 30, and 100 µg/mL) and different alternatingmagnetic field (AMF) exposure times (0, 5, 10, 20, and 30 min) and X-ray irradiation (5 Gy). After 24 hof treatment, (a) cell viability decreased in a concentration- and time-dependent manner, whereas(b) cell death induction increased accordingly. Data are presented as mean ± standard error (SE)from three independent experiments (n = 3).Surgery, chemotherapy, and radiation therapy are the most common cancer treatmentscurrently available, with several new therapies under development [37–39]. Drug deliverysystems (DDS) enhance the therapeutic efficiency of chemotherapy. The MNPs presentedhere can be used in combination with chemoradiotherapy and hyperthermia, in addition todelivering high concentrations of anticancer drugs locally. Furthermore, if several recentlydeveloped chemotherapeutic agents [40,41] can also be encapsulated in these MNPs, itwould be possible to create a therapy that enhances antitumor effects while reducingsystemic side effects. Research is actively being conducted with this goal in mind.This study had several limitations and challenges. The MNPs tended to aggregate duringlayering, as shown by changes in particle size and electron microscopic images. Maintainingan appropriate particle size is crucial for the therapeutic effect of DDS [42]. A particle designthat prevents aggregation as the number of layers increases is needed to ensure the particle sizeMolecules 2025, 30, 1382 9 of 13matches the tumor characteristics. Additionally, to load more anticancer agents, a techniquefor coating multiple layers of anticancer drugs must be developed.These results suggest that the combination of hyperthermia therapy and radiationtherapy using LBL MNPs could be a novel treatment strategy for intractable tumors suchas pancreatic cancer. Optimizing the concentration of LBL MNPs and the duration of AMFexposure may maximize the therapeutic effect and minimize side effects. In the future,in vivo efficacy verification and safety evaluations are expected to be promoted, paving theway for further clinical application. The results of this study indicate that the combinationof LBL MNPs, AMF, and radiation therapy provides a powerful therapeutic effect on tumorcells and is gaining attention as a new option for future cancer treatment.3. Materials and Methods3.1. Preparation of LBL Nanoparticles and LBL Nanoparticles Loaded with GEMThe LBL nanoparticles were prepared using electrostatic adsorption. Briefly, positivelycharged MNPs (EMG 607, Ferrotec Material Technologies Corporation, Tokyo, Japan) andnegatively charged PSS (5 mg/mL, 516-85751, FUJIFILM Wako Pure Chemical Corporation,Osaka, Japan) were mixed at a ratio of 1:49 for 15 min, placed in a neodymium magnetfor 10 min, and centrifuged at 4 ◦C (15,000 rpm, 15,850 rcf) for 15 min to remove thesupernatant. The precipitate was washed with Milli-Q water and centrifuged twice toremove unbound PSS, obtaining the first layer of LBL nanoparticles. Subsequently, theprecipitate was resuspended in a small amount of double-distilled water, shaken, andmixed with PAH (5 mg/mL, 283223, Merck KGaA, Darmstadt, Germany) at a ratio of 1:40for 15 min. As described above, after washing with Milli-Q water and centrifuging twice,the solution was repeatedly mixed with the PSS solution to prepare LBL MNPs with adifferent number of layers.For LBL MNPs loaded with GEM (G-0367-1G, Tokyo Chemical Industry Co., Ltd.,Tokyo, Japan) after preparing the seventh layer of LBL MNPs, negatively charged LBLMNPs and positively charged GEM were electrostatically adsorbed to prepare LBL MNPscontaining GEM. Specifically, GEM (5 mg/mL) and LBL MNPs were mixed, shaken for15 min, and then centrifuged at 15,000 rpm (15,850 rcf) for 15 min at 4 ◦C to remove unboundGEM. The mixture was washed with Milli-Q water and centrifuged twice (15,000 rpm,15,850 rcf, 15 min, 4 ◦C) to obtain LBL MNPs containing GEM.3.2. Characterization of LBL Nanoparticle and Size Distribution and Zeta Potential MeasurementsThe particle size and zeta potential of the LBL nanoparticles were measured usingdynamic light scattering (DLS; Brookhaven Instrument Corp., Holtsville, NY, USA) anda Zetasizer (Malvern Instrument Ltd., Worcestershire, UK), respectively. All particle sizemeasurements were performed using a He-Ne laser beam at 658 nm and a scattering angleof 90◦. The final micelle concentration was 1 mg/mL. For each sample, the data obtainedfrom three measurements were averaged to yield the mean particle size and zeta potential.3.3. Heat Generation Behavior of LBL MNPs upon AMFThe heating profiles of LBL MNPs with and without were investigated by placingthem in the center of a copper coil and applying an AMF (HOSHOT2, Alonics Co., Ltd.,Tokyo, Japan). Heat was generated by the AMF (1.1 kA/m, 281 kHz, 4.0 V). The heatingprofiles were recorded by taking photos every 20 s using a FLIR thermal camera (CPA-E6,FLIR systems Japan K.K., Tokyo, Japan).Molecules 2025, 30, 1382 10 of 133.4. Cell CultureThe human pancreatic cell line PANC-1 (RCB2095; RIKEN Bioresearch Center, Tsukuba,Japan) was cultured in Ham’s F-12 medium (087-08335, FUJIFILM Wako Pure ChemicalCorporation, Osaka, Japan) containing 10% fetal bovine serum (FBS; Lot. 2404079, ThermoFisher Scientific K.K., Tokyo, Japan) and antibiotics (100 U/mL penicillin and 100 µg/mLstreptomycin; Merck KGaA, Darmstadt, Germany) in a 5% CO2 incubator at 37 ◦C. Cellswere passaged twice per week, and treatment samples were prepared three days beforeAMF treatment.3.5. Cytotoxic Effects of LBL MNPs AloneThe cytotoxicity of LBL MNPs alone was evaluated using 5-, 7-, 9-, and 11-layer LBLMNPs. PANC-1 cells were seeded at 1 × 104 cells/500 µL/well in a 24-well plate (3526,Corning, NY, USA) two days before LBL MNP treatment. The following day, the cellswere treated with LBL MNPs at concentrations of 0, 1, 3, 10, 30, and 100 µg/mL for 24 h.After 24 h, cell viability was assessed using the CellTiter-Glo 2.0 assay (G9241, Promega,Madison, WI, USA) according to the manufacturer’s protocol. The assays were conductedin triplicate, and luminescence signals were measured using a microplate reader (Infinite200 PRO, TECAN, Männedorf, Switzerland). To minimize potential interference fromLBL MNPs in fluorescence-based assays, the medium was replaced with a fresh culturemedium before measurement. Specifically, after 24 h of incubation with LBL MNPs, theculture medium containing the nanoparticles was removed, and the cells were washed withphosphate-buffered saline (PBS) (166-23555, FUJIFILM Wako Pure Chemical Corporation,Osaka, Japan). Fresh culture medium was then added before performing cell viability andcytotoxicity assays. This procedure ensured that the fluorescence signals were not affectedby nanoparticle-induced scattering or absorption effects.3.6. Cytotoxic Effects Using LBL Nanoparticles Combined with AMF and/or X-RaysUsing the 7-layer LBL MNPs, which were the most effective in the previous experiment,we evaluated the cytotoxicity of LBL MNPs combined with AMF and X-rays. Two daysbefore LBL MNPs treatment, PANC-1 cells were seeded at 2 × 104 cells/300 µL/well in aµ-Dish 35 mm Quad (80416, ibidi GmbH, Gräfelfing, Germany) with four seeding areas.The cells were treated with 7-layer LBL MNPs at concentrations of 0, 2, 10, and 50 µg/mLfor 24 h. The cells were then subjected to AMF (1.1 kA/m, 281, and 4.0 V) for 0, 10, and30 min. The cells were subsequently irradiated with 5 Gy X-rays at 130 kV, 5 mA, a doserate of 0.5 Gy/min, and room temperature using an X-ray generator RX-650 (FaxitronBioptics LLC, Tucson, AZ, USA) installed at the Proton Medical Research Center, Universityof Tsukuba. Twenty-four hours after irradiation, cell proliferation activity was evaluatedusing Celltiter-Glo 2.0 (G9241, Promega, Madison, WI, USA), and changes in the number ofdead cells were evaluated using the CellTox Green Cytotoxicity Assay (G8742, Promega,Madison, WI, USA) according to the manufacturer’s protocol. The assays were conductedin triplicate, and luminescence signals were measured using a microplate reader (Infinite200 PRO, TECAN, Männedorf, Switzerland). Regarding cell proliferation activity and theincrease in the number of dead cells, the relative light unit (RLU) values obtained fromthe plate reader were normalized with the LBL MNPs concentration of 0 µg/mL and AMFapplication time of 0 min as the standard.3.7. Statistical AnalysisAll statistical analyses were performed using IBM SPSS Statistics (version 29.0.2.0,IBM Corp., Armonk, NY, USA). Data are expressed as the mean ± standard error of themean (SE) obtained from three independent experiments. A two- or three-way ANOVAMolecules 2025, 30, 1382 11 of 13was conducted to assess the effects of layer, concentration, and X-ray exposure on cellviability. Interaction effects were also analyzed. If significant effects were detected, multiplecomparisons were performed using the Bonferroni correction to control for Type I error. Ap-value of < 0.05 was considered statistically significant.4. ConclusionsLBL MNPs have been developed for combination chemotherapy, thermotherapy, andRT. To deliver the anticancer drug GEM, the surface charge of the MNPs was controlledthrough the electrostatic interaction between PSS and PAHs, which was designed to bindGEM to the outermost shell. When the cytotoxicity of GEM-loaded LBL MNPs was ex-amined, particles with 7 layers of PSS/PAHs showed the most effective cytotoxicity. Thisis because the surface charge is precisely controlled by the LBL, and GEM is releasedgradually through exchange with the body’s electrolytes. In addition, AMF irradiationwas found to induce fever, and the combination of chemotherapy, hyperthermia, and RTshowed synergistic cytotoxicity. This may be because hyperthermia and RT complementeach other’s limitations, and GEM is most effective when the cell cycle is arrested in the Sphase, where DNA damage is difficult to repair [24,26]. Beyond conventional chemotherapyand RT, the delivery of drugs and heat sources that synergize these treatments is expectedto drive the development of more efficient combination therapies.Author Contributions: Conceptualization, N.F. (Nobuyoshi Fukumitsu), Y.M. and M.E.; methodology,Y.M. and L.C.; software, Y.M. and L.C.; validation, Y.M., L.C. and N.F. (Nanami Fujisawa); formalanalysis, Y.M. and L.C.; investigation, Y.M., L.C., Y.S., N.F. (Nanami Fujisawa), E.N., S.O. and E.O.;resources, N.F. (Nanami Fujisawa) and M.E.; data curation, N.F. (Nobuyoshi Fukumitsu), Y.M., L.C.and M.E.; writing—original draft preparation, N.F. (Nobuyoshi Fukumitsu), Y.M., L.C. and M.E.;writing—review and editing, N.F. (Nobuyoshi Fukumitsu), Y.M., L.C. and M.E.; visualization, N.F.(Nobuyoshi Fukumitsu), Y.M., L.C. and M.E.; supervision, N.F. (Nobuyoshi Fukumitsu), Y.M. andM.E.; project administration, N.F. (Nobuyoshi Fukumitsu), Y.M. and M.E.; funding acquisition, Y.M.and T.S. All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by JSPS KAKENHI (Grant Numbers JP15K19768 and JP17K15016)to T.S. and the Tsukuba Innovation Arena (TIA) Collaborative Program Exploration and PromotionProject Kakehashi (Grant Number TK20-044) to Y.M.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: The data supporting the findings of this article are available from thecorresponding author, Y.M., upon reasonable request.Acknowledgments: We acknowledge the exceptional work of the Proton Medical Research Center atthe University of Tsukuba in radiobiology.Conflicts of Interest: The authors declare no conflicts of interest.AbbreviationsThe following abbreviations are used in this manuscript:LBL Layer-by LayerGEM GemcitabineMNP Magnetic NanoparticleEPR Enhanced Permeability and RetentionDLS Dynamic Light ScatteringPDI Polydispersity IndexPSS Poly (sodium 4-styrenesulfonate)Molecules 2025, 30, 1382 12 of 13PAH Poly (Allylamine hydrochloride)TEM Transmission Electron MicroscopyAMF Alternated Magnetic FieldROS Reactive Light ScatteringRT RadiotherapyDDS Drug Delivery SystemFBS Fetal Bovine SerumPBS Phosphate-Buffered SalineRLU Relative Light UnitsReferences1. 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MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.https://doi.org/10.1126/science.277.5330.1232https://doi.org/10.1039/D1CP04669Ahttps://doi.org/10.1002/jps.22006https://doi.org/10.1155/2017/1047697https://doi.org/10.1039/c3tb21193bhttps://www.ncbi.nlm.nih.gov/pubmed/32261055https://doi.org/10.1080/10717544.2019.1709922https://doi.org/10.1002/biot.201100045https://doi.org/10.2174/1871520616666161031143301https://doi.org/10.3109/02656736.2013.837200https://doi.org/10.3109/02656731003745740https://doi.org/10.3390/mi13081279https://doi.org/10.1039/D0TB00364Fhttps://doi.org/10.7150/thno.21297https://doi.org/10.1634/theoncologist.10-1-34https://doi.org/10.3390/genes10010025https://doi.org/10.1016/j.addr.2011.03.008https://doi.org/10.1021/acsnanoscienceau.3c00041https://www.ncbi.nlm.nih.gov/pubmed/38644966https://doi.org/10.1016/j.lfs.2022.120729https://www.ncbi.nlm.nih.gov/pubmed/35753439https://doi.org/10.3390/nano8060401https://www.ncbi.nlm.nih.gov/pubmed/29865277https://doi.org/10.3390/cancers3033279https://doi.org/10.18632/oncotarget.16723https://doi.org/10.1007/s11033-023-08809-3https://doi.org/10.1056/NEJMoa1304369https://doi.org/10.1159/000439171https://doi.org/10.1038/nnano.2011.166https://www.ncbi.nlm.nih.gov/pubmed/22020122 Introduction  Results and Discussion  Characterization of the Particle Size and Potential of LBL MNPs  Heat Generation Behavior of LBL MNPs  Relationship Between Cell Killing Effect of LBL MNPs, Concentration, and Layer Number  Enhancement of the Synergistic Cell-Killing Effect by LBL MNPs and AMF Application  Enhancement of the Synergistic Cell-Killing Effect by LBL MNPs, AMF, and X-Ray Irradiation  Materials and Methods  Preparation of LBL Nanoparticles and LBL Nanoparticles Loaded with GEM  Characterization of LBL Nanoparticle and Size Distribution and Zeta Potential Measurements  Heat Generation Behavior of LBL MNPs upon AMF  Cell Culture  Cytotoxic Effects of LBL MNPs Alone  Cytotoxic Effects Using LBL Nanoparticles Combined with AMF and/or X-Rays  Statistical Analysis  Conclusions  References