Influence of hydrogel and porous scaffold on the magnetic thermal property and anticancer effect of Fe 3 O 4 nanoparticles

Influence of hydrogel and porous scaffold on the magnetic thermal property and anticancer effect

deliver MNPs could affect their performance, and appropriate matrices should be designed to maximize their therapeutic effect for biomedical applications.Keywords: Fe 3 O 4 NPs, hydrogel, scaffold, agarose, gelatin, magnetic hyperthermia, anticancer

INTRODUCTION
Magnetic nanoparticles (MNPs) can convert magnetic energy to thermal energy when subjected to alternating magnetic field (AMF) [1,2] .The heat generated by MNPs under AMF irradiation increases the local temperature to ablate cancer cells, known as magnetic hyperthermia (MH) [3][4][5] .MH has been developed as an effective approach for cancer treatment due to its good biocompatibility and deep strong tissue penetration.This approach has also been combined with radiotherapy, chemotherapy, and immunotherapy to further improve the anticancer effect [6][7][8][9][10][11][12] .It is pivotal to increase magnetic-thermal conversion efficiency to achieve a maximized therapeutic effect with the minimized dosage of MNPs.
To achieve high magnetic-thermal conversion efficiency, many studies have reported the optimization of synthesis methods of MNPs [13,14] by controlling their structure and magnetic characteristics, including shape, size, size distribution, dispersion and aggregation state, crystallinity, composition, and magnetic parameters [15][16][17][18][19][20][21][22][23][24][25][26] .Except for the intrinsic properties of MNPs, their surrounding microenvironmental matrices can affect the magnetic-thermal conversion [27,28] .Incorporation of MNPs in hydrogels has been reported to change their magnetic-thermal conversion property [28][29][30][31] .Engelmann et al. immobilized them in acrylamide hydrogels and found that the heating efficiency of MNPs decreased when the hydrogel stiffness increased [28] .Suto et al. compared the influence of polyvinyl alcohol hydrogel and water [31] .The heating efficiency of MNPs dispersed in water was better than that dispersed in polyvinyl alcohol hydrogel, and their specific absorption rate value in hydrogel showed 67% less than that in water [31] .These studies suggest the inhibitory effects of hydrogels on the magnetic-thermal conversion property of MNPs.
In recent years, localized delivery of photothermal nanoparticles (NPs) has been demonstrated as an efficient strategy to accumulate and constrain them in tumors to maximize the photothermal ablation effect while decreasing their side effect [32][33][34][35][36][37] .Both hydrogels and porous scaffolds are good carriers for the local delivery of photothermal NPs.However, the influence of porous scaffolds on the magnetic-thermal conversion of MNPs remains elusive.It is desirable to compare the influence of aqueous solution, hydrogels [38][39][40][41][42][43][44][45] , and porous scaffolds [46][47][48] on the magnetic-thermal conversion of MNPs to maximize their MH effect.
Based on the above considerations, in this study, different microenvironments of phosphate buffer saline (PBS), hydrogels, and porous scaffolds were used to investigate the magnetic-thermal conversion property and anticancer effect of MNPs under AMF irradiation [Figure 1].The same concentration of Fe 3 O 4 NPs was used in PBS, agarose hydrogels, and gelatin porous scaffolds to disclose the influence of the different matrices on magnetic thermal effects.Moreover, Fe

Synthesis and Characterization of Fe 3 O 4 NPs
Fe 3 O 4 NPs were synthesized using Fe (II) and Fe (III) in a mixture solution of DEG and NMDEA (1:1, v/v) [49,50] .First, 4 mmol FeCl 2 •4H 2 O and 2 mmol FeCl 3 •z`6H 2 O were added and dissolved in an 80 mL polyols mixture under a nitrogen atmosphere at room temperature.Next, 40 mL of 400 mM NaOH in a polyol mixture was dropped into the iron salts solution and stirred for 3 h.Then, the mixed solution was heated to 220 °C and reacted for 12 h under the protection of nitrogen.After the reaction, the black precipitates were collected and washed with ethanol/ethyl acetate solvent (1:1, v/v) three times to obtain bare Fe 3 O 4 NPs.The obtained bare Fe 3 O 4 NPs were redispersed in sodium citrate aqueous solution and reacted at 60 °C for 24 h to modify the Fe 3 O 4 NPs with citrate anions.After centrifugation, washing, and lyophilization, the citrate-modified Fe 3 O 4 NPs were obtained.The prepared Fe 3 O 4 NPs were redispersed in PBS to obtain the Fe 3 O 4 NPs colloidal solution at a concentration of 5 mg mL -1 , 10 mg mL -1 , and 20 mg mL -1 , which were named free Fe 3 O 4 -5 (5 mg mL -1 ), free Fe 3 O 4 -10 (10 mg mL -1 ), and free Fe 3 O 4 -20 (20 mg mL -1 ), respectively.The structure and mean diameter of the citrate-modified Fe 3 O 4 NPs were analyzed by a transmission electron microscope (TEM, JEOL 2100F, Japan).The hydrodynamic size of the citratemodified Fe 3 O 4 NPs was analyzed by dynamic light scattering (DLS, Beckman Coulter, USA).

Preparation and characterization of agarose/Fe 3 O 4 hydrogels and gelatin/Fe 3 O 4 scaffolds
The agarose/Fe 3 O 4 hydrogels were prepared by dispersing the citrate-modified Fe 3 O 4 NPs in agarose hydrogels.At first, 0.1 g agarose was dissolved in 5 mL PBS at 110 °C.After the temperature of the agarose solution was cooled to around 40 °C, the citrate-modified Fe 3 O 4 NPs suspension solution was added to prepare 1% agarose solution with Fe 3 O 4 NPs at 5 mg mL -1 , 10 mg mL -1 , and 20 mg mL -1 .Agarose hydrogels without Fe 3 O 4 NPs were prepared as a control.After vortexing, the mixture was added into cylinder molds (Φ10 mm × H4 mm) and immediately transformed into a 4 °C refrigerator for 30 min to form agarose/Fe 3 O 4 hydrogels.The obtained hydrogels with different NP concentrations were referred to as agarose/Fe 3 O 4 -5 (5 mg mL -1 ), agarose/Fe 3 O 4 -10 (10 mg mL -1 ), and agarose/Fe 3 O 4 -20 (20 mg mL -1 ).

Magnetic thermal property of Fe 3 O 4 NPs in different matrices
The magnetic thermal properties of free Fe 3 O 4 NPs, agarose/Fe 3 O 4 hydrogels, and gelatin/Fe 3 O 4 porous scaffolds were investigated under AMF irradiation.First, 300 μL of free Fe 3 O 4 -5, Fe 3 O 4 -10, and Fe 3 O 4 -20 solutions were added in 0.5 mL Eppendorf tubes, respectively.The samples were placed in the center of Double H CoilSets AMF (Frequency: 373.6 kHz; Field intensity: 130 Gauss) for 10 min using a D5 series machine (nB nanoScale BioMagnetics, Zaragoza, Spain).Subsequently, the IR1 thermal imaging system (nB nanoScale Biomagnetics, Zaragoza, Spain) was used to record the temperature change of different samples.Triplicate samples were used for each measurement.
Similarly, 300 μL of aqueous agarose solution without or with 5 mg mL -1 , 10 mg mL -1 , and 20 mg mL -1 of Fe 3 O 4 NPs were added in 0.5 mL Eppendorf tubes, respectively.All samples were immediately transformed into a 4 °C refrigerator for 30 min to form agarose hydrogel, agarose/Fe 3 O 4 -5, agarose/Fe 3 O 4 -10, and agarose/Fe 3 O 4 -20 hydrogel samples.Before exposure to AMF, the agarose/Fe 3 O 4 hydrogel samples were balanced at room temperature for 2 h.The temperature change of the agarose/Fe 3 O 4 hydrogel samples was measured and recorded during 10 min AMF irradiation at 373.6 kHz of frequency and a field intensity of 130 Gauss.Triplicate samples were used for each measurement.
The gelatin/Fe 3 O 4 porous scaffolds were molded into cylinder discs (Φ10 mm × H4 mm) and hydrated with pure water (300 μL/disc) in silicone frames.The gelatin/Fe 3 O 4 porous scaffold discs were exposed to AMF for 10 min (Frequency: 373.6 kHz; Field intensity: 130 Gauss), and the temperature change was measured.
Triplicate samples were used for each measurement.

Anticancer effect of Fe 3 O 4 NPs in different matrices
The anticancer effect of free Fe 3 O 4 NPs, agarose/Fe 3 O 4 hydrogels, and gelatin/Fe 3 O 4 porous scaffolds was investigated by incubating breast cancer cells in the different matrices under AMF irradiation [55][56][57][58][59][60][61] .Three culture modes were used to simulate the cells directly adhered to, near, or far away from the matrices [Figure 1].The cells were seeded in wells of cell culture plates, and then free Fe 3 O 4 NPs, agarose/Fe 3 O 4 hydrogels, or gelatin/Fe 3 O 4 porous scaffolds were added to the adhered cells.The hydrogels and porous scaffolds were sitting on the adhered cells (sitting mode), which simulated the cells near the matrices.The second mode was a transwell mode by seeding cells in the bottom wells of the transwell plates and placing the hydrogels and porous scaffolds in the inserts, which simulated the cells far away from the matrices.The third mode was an adhesion mode by seeding cells on the hydrogels or in the porous scaffolds to allow the cells to adhere to the hydrogels or in the pores of the porous scaffolds, which simulated the cells directly adhering to the matrices.

Anticancer effect of free Fe 3 O 4 NPs
The free Fe 3 O 4 NPs could only be added in the culture medium.Therefore, only the sitting mode was used for investigating the anticancer effect of free Fe 3 O 4 NPs.The sub-cultured MDA-MB-231-Luc cells were harvested and resuspended in a culture medium at a concentration of 2.5 × 10 5 cells mL −1 .A 200 μL cell suspension solution was seeded in the wells of a 48-well plate (5 × 10 4 cells well -1 ).After culture in a humidified incubator (5% CO 2 , 37 °C) for 24 h, the culture medium was removed, and another 200 μL fresh culture medium was added.Then, 300 μL medium, without or with free Fe 3 O 4 -5, free Fe 3 O 4 -10 and free Fe 3 O 4 -20, was added to the wells, respectively.After co-incubation for 2 h, the wells were exposed to AMF (frequency: 373.6 kHz of; field intensity: 130 Gauss) for 10 min.Cell viability before and after AMF irradiation was visualized by live/dead staining and quantitatively analyzed by WST-1 assay.Triplicate samples were used for each measurement.

Anticancer effect of agarose/Fe 3 O 4 hydrogels
The three culture modes were used for the investigation of the anticancer effect of the agarose/Fe 3 O 4 hydrogel.For the sitting mode, the MDA-MB-231-Luc cells were seeded and cultured in the wells of a 48well plate, as mentioned above.After the culture medium was changed with another 200 μL fresh medium, the agarose and agarose/Fe 3 O 4 hydrogel discs (Φ10 mm × H4 mm) were placed on the cells.After coincubation for 2 h, the wells were exposed to AMF (frequency: 373.6 kHz of; field intensity: 130 Gauss) for 10 min.Cell viability was investigated by live/dead staining and WST-1 assay before and after AMF irradiation.Triplicate samples were used for each measurement.
For the transwell mode, the MDA-MB-231-Luc cells were seeded in the centers of the wells of 24-well plate by using donut-shaped silicone rings (inner diameter 10 mm, outer diameter: 16 mm, height: 2 mm).The seeded cell number was the same (5 × 10 4 cells well -1 ).The agarose/Fe 3 O 4 hydrogel discs were placed on the transwell inserts and co-cultured with the cells on the bottom wells.The transwell plates containing cells and discs were irradiated by AMF (frequency: 373.6 kHz; magnetic field: 130 Gauss) for 10 min.Before and after AMF irradiation, cell viability was investigated, as mentioned above.Triplicate samples were used for each measurement.
For the adhesion mode, the agarose/Fe 3 O 4 hydrogel discs (Φ10 mm × H4 mm) were put in the wells of a 48well plate.Subsequently, 200 μL of cell suspension solution was seeded on the agarose/Fe 3 O 4 hydrogel discs.After 2 h incubation, the plates were exposed to AMF (frequency: 373.6 kHz; magnetic field: 130 Gauss) for 10 min, and cell viability was investigated, as mentioned above.Triplicate samples were used for each measurement.

Anticancer effect of gelatin/Fe 3 O 4 porous scaffolds
The three culture modes were also used for the investigation of the anticancer effect of the gelatin and gelatin/Fe 3 O 4 porous scaffolds.All the experiment procedures were the same as those of agarose/Fe 3 O 4 hydrogel discs by using the porous scaffold discs (Φ10 mm × H4 mm).Triplicate samples were used for each measurement.

Characterization of Fe 3 O 4 NPs
The morphology and size of the citrate-modified Fe 3 O 4 NPs were characterized by TEM.As shown in Figure 2A-C, the NPs displayed a flower-like shape, which should have a good magnetic-thermal conversion capacity for MH.They had an average size of 30.8 ± 5.7 nm from the TEM images.The hydrodynamic size of the citrate-modified Fe 3 O 4 NPs was measured in aqueous solution by DLS, and the hydrodynamic size was 108.5 ± 28.5 nm [Figure 2D].3C].The gelatin and gelatin/Fe 3 O 4 porous scaffolds had the same pore structures.They had large spherical pores that were surrounded by some small pores [Figure 3D].The large spherical pores were controlled by the ice particulates that were used as a porogen material.The results indicated that the embedding of Fe 3 O 4 NPs did not affect the pore structures of hydrogels and porous scaffolds.

Magnetic thermal property of Fe 3 O 4 NPs in different matrices
The magnetic thermal properties of Fe 3 O 4 NPs in PBS, agarose hydrogels, and gelatin porous scaffolds were investigated by applying AMF (frequency: 373.6 kHz; field: 130 Gauss) for 10 min, and the results are shown in Figure 4 and Table 1.The temperature of PBS, agarose hydrogels, and gelatin porous scaffolds without Fe 3 O 4 NPs had no obvious change after AMF irradiation [Figure 4A and Table 1].The results suggested that PBS, agarose hydrogels, and gelatin porous scaffolds had no magnetic-thermal conversion capacity in the absence of Fe      The WST-1 assay showed that the cells cultured without or with free Fe 3 O 4 NPs had the same high viability before AMF irradiation [Figure 5B].After AMF irradiation, the viability of cells cultured without free Fe 3 O 4 NPs did not change significantly, while the viability of cells cultured with free Fe 3 O 4 NPs significantly decreased after AMF irradiation.Viability of cells cultured with 5, 10, and 20 mg mL -1 free Fe 3 O 4 NPs decreased to 10.3% ± 6.9%, 3.1% ± 5.8%, and 2.7% ± 5.2%, respectively.All the live/dead staining and WST-1 assay results indicated that the breast cancer cells could be killed by the free Fe 3 O 4 NPs under AMF irradiation.A higher concentration of free Fe 3 O 4 NPs resulted in a higher killing effect.The killing effect of free Fe 3 O 4 NPs should be due to the high temperature generated by free Fe 3 O 4 NPs during AMF irradiation [Figure 4].A higher concentration of the Fe 3 O 4 NPs could generate higher temperatures and enhance the killing efficiency.

Anticancer effect of agarose/Fe 3 O 4 hydrogels
The interaction between cells and agarose/Fe 3 O 4 hydrogels should be different from that between cells and free Fe 3 O 4 NPs.The cells could be near the hydrogels without adhesion to the hydrogels.The cells could also be far away from the hydrogels or directly adhere to the hydrogels.To simulate these interactions between the breast cancer cells and hydrogels, three culture modes were used to investigate the MH anticancer effect of the agarose/Fe  6D].
The breast cancer cells adhered on the agarose/Fe 3 O 4 hydrogel discs (adhesion mode) were most efficiently killed by AMF irradiation.Almost all the cells cultured with all the agarose/Fe 3 O 4 hydrogel discs were dead, and their viability was the lowest compared to the sitting and transwell modes [Figure 6E and F].Viability of breast cancer cells adhered on the agarose/Fe 3 O 4 -5, agarose/Fe 3 O 4 -10, and agarose/Fe 3 O 4 -20 decreased to 7.1% ± 1.2%, 2.7% ± 4.6%, and 2.3% ± 6.5%, respectively, after AMF irradiation [Figure 6F].This should be due to the direct heating effect of the cells by the agarose/Fe 3 O 4 hydrogels when the cells adhered to the hydrogels.
The above results indicated that the breast cancer cells could be killed by either free Fe 3 O 4 NPs, agarose/Fe 3 O 4 hydrogels, or gelatin/Fe 3 O 4 porous scaffolds.However, the anticancer effect depended on the matrices and interactions.The free Fe 3 O 4 NPs showed the highest killing effect, while the gelatin/Fe 3 O 4 porous scaffolds showed the lowest.Matrices used to embed Fe 3 O 4 NPs showed an inhibitory influence on the killing effect of Fe 3 O 4 NPs.In particular, the porous scaffolds had a more inhibitory influence than the hydrogels.This should be due to the influence of matrices on the magnetic thermal property of the Fe 3 O 4 NPs.The interaction between breast cancer cells and Fe 3 O 4 NP-embedded hydrogels or porous scaffolds  The matrix influence should be due to the variation of Brownian relaxation of Fe 3 O 4 NPs in the matrices.The heat generation mechanism of MNPs exposed to AMF includes Néel relaxation and Brownian relaxation [62] .Néel relaxation refers to the heating due to the energy loss produced by the rotation of individual magnetic moments within the MNPs under AMF irradiation, and Brownian relaxation refers to the rotation of entire MNPs to produce heat [63,64] .The Néel relaxation of Fe The results of this study revealed that the magnetic thermal property and anticancer effect of Fe 3 O 4 NPs were affected by their surrounding microenvironmental matrices.Although cancer cells and normal cells have different cytoplasmic properties [65,66] and cancer cells are more sensitive to heat compared to normal cells [67,68] , the heat generated by Fe 3 O 4 NPs under AMF irradiation should also affect viability of normal cells.The influence of Fe 3 O 4 NP-embedded hydrogels and porous scaffolds on normal cell viability needs further investigation for controlling temperature to efficiently ablate cancer cells while minimizing negative effects on normal cells.
Furthermore, the interaction between the breast cancer cells and the Fe 3 O 4 NPs could affect the killing effect.The free Fe 3 O 4 NPs could be uptaken by breast cancer cells and generate heat inside the cells under AMF irradiation.When the Fe 3 O 4 NP-embedded agarose hydrogels were applied, the breast cancer cells could be adhered to the hydrogel, near the hydrogels, or far away from the hydrogels.For the Fe 3 O 4 NPembedded gelatin porous scaffolds, breast cancers could enter the scaffolds and adhere in the scaffolds, near the scaffolds, or far away from the scaffolds.Three cultured models (sitting, transwell, and adhesion modes) were used to simulate the interaction between the cells and matrices.The Fe 3 O 4 NPs embedded in the agarose hydrogels and gelatin porous scaffolds should be less or not uptaken by the cells if the hydrogels and scaffolds were not degraded.Therefore, the heat should be generated by the scaffolds and then transmitted to the breast cancer cells for ablation.The breast cancer cells adhered to the hydrogels or in the scaffolds were most efficiently ablated.The cells far away from the hydrogels and porous scaffolds were less affected.The results should be due to the heat transmission effect of the hydrogels and porous scaffolds.

CONCLUSION
In this study, the magnetic thermal property and ablation effect of free Fe

Authors' contributions
Made substantial contributions to the conception and design of the study and performed data analysis and interpretation: Wang M, Sun R, Kawazoe N, Chen G Performed data acquisition and provided administrative, technical, and material support: All authors

3 O 4
NPs at different concentrations were incorporated in the same matrix to study the MNP concentration dependence.The free Fe 3 O 4 NPs in PBS exhibited the best magnetic thermal property, while embedding in agarose hydrogels or gelatin porous scaffolds decreased the temperature change during AMF irradiation.The anticancer effect was investigated in vitro by incubating breast cancer cells (MDA-MB-231-Luc cells) with free Fe 3 O 4 NPs, agarose/Fe 3 O 4 hydrogels, and gelatin/Fe 3 O 4 porous scaffolds under AMF irradiation.The free Fe 3 O 4 NPs showed the highest anticancer effect under AMF irradiation, followed by Fe 3 O 4 NPs in agarose hydrogels and then Fe 3 O 4 NPs in gelatin porous scaffolds.The matrices for MNP delivery could affect the magnetic-thermal conversion property and anticancer effect of Fe 3 O 4 NPs.

Figure 1 .
Figure 1.Anticancer experimental scheme of free Fe 3 O 4 NPs (A), agarose/Fe 3 O 4 hydrogels (B-D), and gelatin/Fe 3 O 4 porous scaffolds(E-G).Three modes (sitting, transwell, and adhesion modes) were used to simulate the cells near or far away from or directly adhered to the matrices.
Characterization of agarose/Fe 3 O 4 hydrogels and gelatin/Fe 3 O 4 porous scaffolds The agarose/Fe 3 O 4 hydrogel discs are shown in Figure 3A.As the concentration of Fe 3 O 4 NPs increased, the appearance of agarose/Fe 3 O 4 hydrogel changed from transparent to black.SEM observation of the lyophilized agarose/Fe 3 O 4 hydrogel discs showed that the agarose hydrogels with different amounts of Fe 3 O 4 NPs had similar structures [Figure 3B].They had spindle-shaped pores.The gelatin porous scaffold without Fe 3 O 4 NPs was white, while the gelatin/Fe 3 O 4 porous scaffolds became gray (gelatin/Fe 3 O 4 -5), dark gray (gelatin/Fe 3 O 4 -10), and black (gelatin/Fe 3 O 4 -20) [Figure

Figure 3 .
Figure 3. Gross appearances (A) and SEM images (B) of agarose and agarose/Fe 3 O 4 hydrogels with different concentrations of citratemodified Fe 3 O 4 NPs.Gross appearances (C) and SEM images (D) of gelatin and gelatin/Fe 3 O 4 porous scaffold with different concentrations of citrate-modified Fe 3 O 4 NPs.

Figure 5 .
Figure 5. Anticancer effect of free Fe 3 O 4 NPs.Live/dead staining of MDA-MB-231-Luc cells cultured without or with free Fe 3 O 4 NPs before and after AMF irradiation (live cell: green fluorescence, dead cells: red fluorescence) (A).Quantified cell viability during culture without or with free Fe 3 O 4 NPs before and after AMF irradiation (B).Cell viability was normalized to that cultured with PBS without free Fe 3 O 4 NPs before AMF irradiation.Data are the mean ± SD (n = 3).Significant difference: ***P < 0.001.N.S. : no significant difference.

3 O 4
NPs and Fe 3 O 4 NPs embedded in agarose hydrogels and gelatin porous scaffolds were investigated to elucidate the influence of microenvironmental matrices on these properties.The flower-like Fe 3 O 4 NPs were embedded in agarose hydrogels and gelatin porous scaffolds.Their magnetic thermal property and anticancer effects were compared with those of free Fe 3 O 4 NPs.Under AMF irradiation, the free Fe 3 O 4 NPs showed the highest temperature increase.Embedding in agarose hydrogels and gelatin porous scaffolds inhibited the heating capacity of Fe 3 O 4 NPs and decreased the temperature change.The gelatin porous scaffolds had the highest inhibitory influence.The anticancer effect of Fe 3 O 4 NPs was also dependent on the matrices.The free Fe 3 O 4 NPs could most efficiently kill breast cancer cells under AMF irradiation.However, the ablation capacity of Fe 3 O 4 NPs embedded in the agarose hydrogels and gelatin porous scaffolds significantly decreased under AMF irradiation compared to that of free Fe 3 O 4 NPs.The reduced killing capacity of Fe 3 O 4 NPs in agarose hydrogels and gelatin porous scaffolds was due to the inhibitory effect of the matrices on their magnetic thermal property.These results suggested that the matrices surrounding MNPs could affect the magnetic thermal property of MNPs and, therefore, affect their ablation capacity to cancer cells.The results should provide useful information for the design and application of MNPs for biomedical applications.

Table 1 . Magnetic thermal property of Fe 3 O 4 NPs in different matrices under AMF irradiation [Mean ± SD (n = 3)] Sample ΔT of PBS (°C) ΔT of agarose hydrogel (°C) Percentage compared to free NPs ΔT of gelatin porous scaffold (°C) Percentage compared to free NPs
3 O 4 hydrogels.When the MDA-MB-231-Luc cells were cultured near the agarose/Fe 3 O 4 hydrogel discs (sitting mode), most of the cells near the agarose/Fe 3 O 4 -5 hydrogel discs were dead, and cell viability decreased significantly after AMF irradiation [Figure 6A and B].Almost all the cells near the agarose/Fe 3 O 4 -10 and agarose/Fe 3 O 4 -20 were dead, and their viability further decreased to the lowest level.The cell viability near agarose/Fe 3 O 4 -5, agarose/Fe 3 O 4 -10, and agarose/Fe 3 O 4 -20 decreased to 11.3% ± 4.8%, 3.9% ± 2.2%, and 3.6% ± 5.1%, respectively, after AMF irradiation [Figure 6B].However, the cells near the agarose hydrogel discs without Fe 3 O 4 NPs were alive, and their viability did not change before and after AMF irradiation.When the breast cancer cells were cultured far away from the agarose/Fe 3 O 4 hydrogel discs (transwell mode), the agarose/Fe 3 O 4 hydrogels with a high concentration of Fe 3 O 4 NPs (agarose/Fe 3 O 4 -10 and agarose/Fe 3 O 4 -20) could kill almost all the cells and significantly decrease cell viability to very low level by AMF irradiation [Figure 6C and D].However, only a small number of cells cultured with the agarose/Fe 3 O 4 -5 were killed by AMF irradiation.After AMF irradiation, cell viability in the transwell of agarose/Fe 3 O 4 -5, agarose/Fe 3 O 4 -10, and agarose/Fe 3 O 4 -20 decreased to 85.7% ± 5.7%, 6.0% ± 3.5% and 2.8% ± 5.9%, respectively [Figure 3 O 4 NPs in the hydrogels and porous scaffolds might not change.However, the matrices should affect the Brownian relaxation of Fe 3 O 4 NPs.The Brownian relaxation of Fe 3 O 4 NPs under AMF irradiation should be partially suppressed in the agarose hydrogel, leading to a decreased heating effect of Fe 3 O 4 NPs.When the Fe 3 O 4 NPs were embedded in gelatin porous scaffolds, the Fe 3 O 4 NPs were tightly constrained in the gelatin fibers, and the Brownian relaxation of Fe 3 O 4 NPs should be heavily inhibited.Therefore, the Fe 3 O 4 NPs in the gelatin porous scaffolds generated heat predominantly through Néel relaxation.Due to the influence of matrices on the magnetic thermal property of Fe 3 O 4 NPs, the anticancer effect of Fe 3 O 4 NPs was also dependent on the matrices.The breast cancer cells cultured with free Fe 3 O 4 NPs were most efficiently killed by AMF irradiation.Embedding in agarose hydrogels and gelatin porous scaffolds significantly decreased the killing effect of Fe 3 O 4 NPs.Embedding in gelatin porous scaffolds showed the lowest killing effect.The decreased anticancer effects of Fe 3 O 4 NPs embedded in agarose hydrogels and gelatin porous scaffolds could be explained by the decreased temperature change under AMF irradiation.