Tailoring mSiO2-SmCox nanoplatforms for magnetic/photothermal effect-induced hyperthermia therapy

Hyperthermia therapy is a hotspot because of its minimally invasive treatment process and strong targeting effect. Herein, a synergistic magnetic and photothermal therapeutic nanoplatform is rationally constructed. The well-dispersive mSiO2-SmCox nanoparticles (NPs) were synthesized through a one-step procedure with the regulated theoretical molar ratio of Sm/Co among 1:1, 1:2, and 1:4 for controlling the dispersion and magnetism properties of SmCox NPs in situ growth in the pore structure of mesoporous SiO2 (mSiO2), where mSiO2 with diverse porous structures and high specific surface areas serving for locating the permanent magnetic SmCox NPs. The mSiO2-SmCox (Sm/Co = 1:2) NPs with highly dispersed and uniform morphology has an average diameter of ∼73.08 nm. The photothermal conversion efficiency of mSiO2-SmCox (Sm/Co = 1:2) NPs was determined to be nearly 41%. The further in vitro and in vivo anti-tumor evaluation of mSiO2-SmCox (Sm/Co = 1:2) NPs present promising potentials for hyperthermia-induced tumor therapy due to magnetic and photothermal effects.


Introduction
Malignant tumors seriously infringe on public health and are the diseases with the highest fatality rate (Tan et al., 2022;Zhang et al., 2022).Abnormal growth and reproduction of tumor cells may spread to tissues and organs throughout the body through the blood and lymphatic system at any time, bringing serious consequences.In recent years, with the improvement of the clinical medical level, remarkable progress has been made in tumor diagnosis and treatment.However, due to the rapid growth and reproduction of tumor cells and ease to spread, the treatment is still a huge challenge.On the other hand, currently widely used traditional treatment methods have great limitations and side effects.Thus, researchers are trying to find new treatments with less toxic side effects and better results.The prosperous development of nanotechnology has provided a new approach for cancer therapeutics, reduced the side effects, and enhanced the targeting efficiency of antitumor drugs (Cai et al., 2019;Chang et al., 2021;Li et al., 2021;Xu and Pu, 2021;Lv et al., 2022;Cao et al., 2023;Li et al., 2023).Significantly, tumor hyperthermia, as a new type of tumor adjuvant therapy, has achieved significant advances (Lv et al., 2017;Liu et al., 2021;Uson et al., 2021;Yu et al., 2021;Chung et al., 2022;Wang et al., 2022).Among them, magnetic hyperthermia therapy and photothermal therapy are becoming hot spots due to their advantages of minimally invasive treatment processes and strong targeting effect (Wu et al., 2019;Zhu et al., 2019;Xu et al., 2020a;Li et al., 2020;Zhang et al., 2020;Zhang X. et al., 2021;Castellanos-Rubio et al., 2021;Wang and Hou, 2021).
Tumor hyperthermia is defined as the method of heating the tumor area to 41-46 °C for treatment, while thermal ablation of the tumor refers to the method of heating the tumor area to more than 56 °C to make the tumor tissue coagulated and necroti.(Murugan et al., 2019;Lima et al., 2021;Lu and Wang, 2021;Xu and Pu, 2021).Tumor cells have poor heat resistance and are prone to apoptosis and necrosis at 40-48 °C, while normal cells and tissues are not affected.Traditional hyperthermia has certain side effects, so it is only used as an auxiliary means of radiotherapy and chemotherapy.In recent years, nanotechnology-induced tumor hyperthermia as a new hyperthermia method has attracted wide attention.Tian et al. reported a near-infrared (NIR)-triggered theranostic nanoplatform (GA-PB@MONs@LA) for synergistic photothermal therapy and enhanced Fenton nanocatalytic therapy against hypoxic tumors (Tian et al., 2022).Magnetic hyperthermia therapy uses the high temperature generated by the magnetic thermal effect, where the magnetic thermal materials in the high-frequency alternating magnetic field (AMF) generate heat to eliminate tumor cells (Beola et al., 2020;Fotukian et al., 2020;Idoia et al., 2020;Qian et al., 2020;Xie et al., 2020;Zhao et al., 2020).Compared with traditional therapies, magnetic hyperthermia therapy is not limited by the depth of treatment and is a non-invasive treatment with strong specificity and targeted effect, which can be used as a sensitizer in combination with chemotherapy, radiation therapy, immunotherapy, and gene therapy to achieve synergistic results.Photothermal therapy uses photothermal agents to transfer light energy into heat energy under light irradiation and release a large amount of heat to ablate tumor cells (Fan et al., 2018;Dalila et al., 2019;Wang et al., 2019;Xu et al., 2019;Zhou et al., 2019;Shi et al., 2021).The high temperature will not only damage the cell membrane of tumor cells and denature proteins but also inhibit the replication of DNA for eliminating tumor cells.Photothermal therapy has the advantages of minimal invasion, low toxicity, and side effects, and high photothermal conversion efficiency, which has great development potential in the field of tumor theranostics (Zeng et al., 2018;Cai et al., 2019;Chen et al., 2019;Deng et al., 2019;Lu et al., 2023).Thus, constructing the "all-in-one" nanoplatforms with both magnetic and photothermal effects for high efficacy of tumor hyperthermia is interesting.
Magnetic nanomaterials with high Curie temperature, high coercivity, good magnetic thermal properties, etc., have a wide range of applications in tumor diagnosis and treatment (Xu et al., 2020b;Wang et al., 2020;Xie et al., 2020).Firstly, they are able to be employed as contrast agents for bio-imaging.Secondly, magnetic nanomaterials gathered inside tumor cells can convert electromagnetic energy into heat energy in the high-frequency AMF and release heat, thus causing tumor cell apoptosis or tumor tissue necrosis (Umut et al., 2019;Chan et al., 2020;Chandrasekharan et al., 2020).Thirdly, the modified magnetic nanomaterials can achieve specific delivery of drugs under the traction of AMF.The magnetic nanomaterials studied at present are mainly iron-based nanomaterials.Interestingly, nanocrystalline permanent magnet materials also show excellent magnetic properties and gradually become a research hotspot in the therapeutic field (Zhang Y. et al., 2021;Wang and Hou, 2021;Chung et al., 2022;Wang et al., 2022).Magnetic materials can be divided into three categories, namely, hard magnetism, semi-hard magnetism, and soft magnetism.The hard magnetic material with high coercivity is not easy to demagnetize after magnetization.After removing the external magnetic field, it can still maintain strong magnetic materials, which is also known as permanent magnet material.So far, rare Earth permanent magnets have gone through three generations, namely, SmCo 5 , Sm 2 Co 17 , and Nd-Fe-B permanent magnet materials.However, controlling the size of permanent magnetic materials in the nanoscale is an important prerequisite for their development in the medical field.Surprisingly, mesoporous silica with diverse porous structures and high specific surface areas can be utilized to locate the permanent magnet nanomaterial separately.
Herein, the well-dispersive mSiO 2 -SmCo x NPs were synthesized through a one-step procedure for magnetic/photothermal effectinduced hyperthermia therapy.The theoretical molar ratio of Sm/Co was systematically regulated among 1:1, 1:2, and 1:4 for controlling the dispersion and magnetism properties of SmCo x NPs in situ growth in the pore structure of mesoporous SiO 2 (mSiO 2 ).The mSiO 2 -SmCo x (Sm/Co = 1:2) NPs with highly dispersed and uniform morphology has an average diameter of ~73.08 nm, and the photothermal conversion efficiency was determined to be nearly 41%.The further in vitro and in vivo anti-tumor evaluation of mSiO 2 -SmCo x (Sm/Co = 1:2) NPs demonstrated promising potentials for hyperthermia-induced tumor therapy due to magnetic and photothermal effects.

Synthesis process
For mSiO 2 -SmCo x NPs, the molar ratio of Sm/Co ions was adjusted as 1:1, 1:2, and 1:4.A fixed amount of Sm(NO 3 ) 3 and the corresponding amount of Co(NO 3 ), as-synthesized mesoporous silicon, and 25 mL of PVP solution (1g mL -1 , in TEG) were mixed through magnetic stirring.Under the vacuum condition, the mixture was heated to 120 °C.Later, a special amount of acetic acid was added and continuously stirred (300 rpm) for 20 min.Next, the mixture was slowly heated to 260 °C at a constant speed (5 °C min -1 ) under the protection of N 2 atmosphere and reacted for another 2 h.After naturally cooling to 25 °C, the product was pretreated by centrifugation treatment (12000 rpm, 10 min) and washed with cyclohexane and ethanol for four times.The mSiO 2 -SmCo x NPs were then obtained after drying overnight (80 °C).

Characterizations
The transmission electron microscopy (TEM, containing EDS and mapping) images were measured on the Tecnai T20 microscope with an operating voltage of 200 kV.The XRD patterns were explored on a DMAX-2400 diffractometer with Cu Kα radiation under 40 kV.X-ray photoelectron spectroscopy (XPS) analysis was operated on a Thermo Scientific K-Alpha spectroscope.Inductively coupled plasma optical emission spectrometer (ICP-OES) was obtained by iCAP 6000 series spectrometry.Ultraviolet-Near Infrared (UV-NIR) absorbance value was tested by a TU-1601 spectrophotometer.The fluorescence intensity of cells and stained tissue sections were obtained with confocal irradiation scanning microscopy TCS SP8.Apoptosis data were characterized by flow cytometry.

Magnetic/photothermal properties
Different concentration of mSiO 2 -SmCo x (Sm/Co = 1:2) NPs solution was placed in a 1.5 mL tube, illuminated with 808 nm laser (1.0 W cm −2 ) for 800 s or imposed on AMF condition.The mSiO 2 -SmCo x (Sm/Co = 1:2) NPs solution was placed in the middle of the coils of an in-house-built magnetic hyperthermia device (coil diameter: 10 cm, frequency: 513 kHz, output power: 8 kW, output current: 28.2 A, output voltage: 361 V).Then, the photograph was captured by an Infrared thermal camera.Besides, the PBS solution as control was irradiated in the same way.

In vitro experiments
The cell lines (L929 and 4T1) present in this study were obtained from FDCC (Ruilu in Shanghai, China).The cell strains were cultivated at 37 °C under 5% CO 2 .Firstly.The confocal laser scanning microscope (CLSM) images were measured to investigate the cell phagocytosis in 4T1 cells.Followed by setting in a cell culture dish with 28 mm cover glass, The 4T1 cells (1 × 10 5 per well) were incubated overnight.Then, DMEM solution loading FITC-labeled mSiO 2 -SmCo x (Sm/Co = 1:2) NPs (1 mL, 50 μg mL -1 ) were added in different time nodes (1, 2, and 3 h).Afterward, an MTT assay was processed to estimate the biocompatibility and cytotoxicity of mSiO 2 -SmCo x (Sm/Co = 1:2) NPs using 4T1 and L929 cells with different concentrations and conditions.With the injection of MTT solution (200 μL, 1 mg mL -1 ) into 4T1 cells for 4 h, the corresponding absorbance was measured to figure out the cell viability.As the above cell treatment, the 4T1 cells were subjected to live/dead cell staining and cultured for 12 h.Then, the cells in each well were treated with different groups and stained by calcein-AM/ PI (4 μM and 8 Μm, respectively) for CLSM observation.For quantitative analysis of cell death, the collecting cells were treated with an annexin V-FITC/PI dual-staining apoptosis detection kit for flow cytometry examination.

Statistical analysis
The results are presented as mean ± S.D. Error bars are dependent on the standard errors of the mean (n = 5).Statistical analysis is presented by the Student's two-sided t-test.*p < 0.05, **p < 0.01, or ***p < 0.001.

Synthesis and characterization
The mSiO 2 -SmCo x NPs were simply synthesized through a onestep procedure, where the SmCo x NPs were in situ grown in the pore structure of mSiO 2 .The theoretical molar ratio of Sm/Co was regulated among 1:1, 1:2, and 1:4 for controlling the dispersion and magnetism properties of mSiO 2 -SmCo x NPs.As exhibited in Figures 1A-E, the TEM images display the nanostructure of SmCo x NPs, mSiO 2 , and mSiO 2 -SmCo x NPs (Sm/Co = 1:1, 1:2, and 1:4).For SmCo x NPs without mSiO 2 (Figure 1A), the free-grown SmCo x NPs are exhibited in the aggregated performance due to the magnetic properties of SmCo x NPs, which are excluded for further bioapplication.The pure mSiO 2 TEM image in Figure 1B demonstrates the well-dispersive manners and the large amount of pore structure.For mSiO 2 -SmCo x (Sm/Co = 1:1 and 1:2) NPs (Figures 1C, D), a uniform spherical morphology with highly monodisperse is observed.The corresponding Sm, Co, Si, and O elements mappings demonstrate the loading and separation functionalities of mSiO 2 nanocarriers and also demonstrate the collective of Co and Sm elements.For mSiO 2 -SmCo x (Sm/Co = 1:4) NPs (Figure 1E), the SmCo x NPs in the pore structure of mSiO 2 show an enhanced aggregated manner.The X-ray diffraction (XRD) patterns of mSiO 2 and mSiO 2 -SmCo x (Sm/Co = 1:2 and 1:4) NPs are shown in Figure 1F, from which the characteristic diffraction peaks could be well indexed to the SmCo 5 (JCPDS No. 35-1368) and mSiO 2 (JCPDS No. 47-1144), respectively, demonstrating the successful formation of magnetic nanoparticles of SmCo 5 .The elemental composition of mSiO 2 and mSiO 2 -SmCo x (Sm/Co = 1: 2 and 1:4) NPs also reveals by energy-dispersive spectroscopy (EDS) mapping images (Figures 2A-C).The zeta potential is explored for mSiO 2 , mSiO 2 -SmCo x (Sm/Co = 1:2) NPs, and PEG-modified mSiO 2 -SmCo x (Sm/Co = 1:2) NPs, changing from 15.3 eV, 12.6 eV, to 19.2 eV, illustrating the good biocompatibility (Figure 2D).The particle size distribution patterns indicate that the mSiO 2 , mSiO 2 -SmCo x (Sm/Co = 1:2) NPs, and PEG-modified mSiO 2 -SmCo x (Sm/Co = 1:2) NPs with high dispersity and uniform morphology have an average diameter of 72.50 73.08, and 76.00 nm, respectively (Figures 2E-G).Furtherly, the element composition is again demonstrated by XPS.The full-scan XPS confirms the existence of Sm 3d, Co 2p, Si 2p, and O 1s in mSiO 2 -SmCo x (Sm/Co = 1:2) NPs (Figure 2H).The binding energy peaks of Si 2p are given in Figure 2I for the form of Si in mSiO 2 .Through peak separation.The high-resolution XPS spectrum of O (Figure 2J) shows the banding energy of 530.8 eV.The high-resolution spectra of Sm 3d at a peak of 1083.5 eV are attributed to Sm 3d5/2 (Figure 1K).From the high-resolution of Co 3d (Figure 2L), two peaks at 781.6 eV and 796.8 eV are attributed to Co 2p 3/2 and Co 2p 1/2 , respectively.The above structure and morphology analysis demonstrate that the mSiO 2 -SmCo x NPs with proper Sm/Co molar ratio equip with promising potentials for further bio-application.

Magnetic/photothermal effects evaluation
Magnetic hyperthermia therapy and photothermal therapy are becoming widespread hotspots due to their advantages of minimally invasive treatment processes and strong targeting effect.Herein, the magnetic and photothermal effects were explored in detail.Compared with optical, acoustic, and electrical fields, the magnetic field shows properties with large force output, high precision, and especially deep tissue penetration.Firstly, the hyperthermia induced by SmCo x NPs and mSiO 2 -SmCo x (Sm/ Co = 1:2 and 1:4) NPs under AFM was characterized.The pure SmCo x NPs group shows the highest temperature increase compared with the mSiO 2 -SmCo x (Sm/Co = 1:2 and 1:4) NPs.Among the mSiO 2 -SmCo x NPs, the hyperthermia-induced performance of mSiO 2 -SmCo x (Sm/Co = 1:4) NPs is better than mSiO 2 -SmCo x (Sm/Co = 1:2) NPs (Figure 3A).The corresponding infrared thermal images of mSiO 2 -SmCo x (Sm/Co = 1:2 and 1:4) NPs directly demonstrate the above result (Figure 3B).However, the aggregated performance of mSiO 2 -SmCo x (Sm/Co = 1:4) NPs is not suitable for bio-application.Thus, according to the structural characterization and magnetic effect property, the mSiO 2 -SmCo x (Sm/Co = 1:2) NPs are chosen for further investigation.

In vivo evaluation
Considering the in vitro high anti-tumor efficiency of mSiO 2 -SmCo x (Sm/Co = 1:2) NPs, we injected mSiO 2 -SmCo x (Sm/Co = 1:2) NPs into 4T1-bearing female BALB/c mice to evaluate the further in vivo antitumor therapeutic performance.Firstly, to ensure biosafety before the therapeutic process, the biological distribution of mSiO 2 -SmCo x (Sm/Co = 1:2) NPs after entering the tumor and organisms were estimated.For confirmed periods, the 4T1 tumor-bearing mice were euthanized after intravenous administrated, then collected the tumor and five main organs for investigating Sm ions concentration to build the biodistribution via ICP-OES analysis (Figure 6A).As being uptaken by the reticuloendothelial system, the mSiO 2 -SmCo x (Sm/ Co = 1:2) NPs mainly enrich in the liver and spleen.The accumulation of mSiO 2 -SmCo x (Sm/Co = 1:2) NPs in tumors are due to the enhanced permeability and retention effect (EPR) effect, where a maximum tumor uptake of the administration around 6 h, demonstrating that mSiO 2 -SmCo x (Sm/Co = 1:2) NPs exhibit a good tumor-targeted administration.Next, the enhanced anticancer effect of mSiO 2 -SmCo x (Sm/Co = 1:2) NPs was explored according to the standard treatment process.When the tumor volume reached nearly 30 mm 3 , the 4T1 tumor-bearing mice were divided into four groups randomly (n = 5 per group): 1) control (PBS), 2) mSiO 2 -SmCo x (Sm/Co = 1:2) NPs, 3) mSiO 2 -SmCo x (Sm/ Co = 1:2) NPs + NIR (1.0 W cm −2 , 10 min), and 4) mSiO 2 -SmCo x (Sm/Co = 1:4) NPs + NIR (1.0 W cm −2 , 10 min) + M (AFM).mSiO 2 -SmCo x (Sm/Co = 1:2) NPs were injected intravenously into each group on 1, 7, and 14 days.All the samples were exposed to NIR irradiation or magnetic conditions after 6 h intravenously injection.The tumor volume and weight of each sample were measured and recorded every 2 days throughout the whole therapy.Moreover, there was no significant weight difference among the groups of mice after 14 days (Figure 6B).As exhibited in Figure 6C, the relative tumor volume displays a trend of differentiation.The tumor growth in the 3) and 4) groups are all suppressed, and the mSiO 2 -SmCo x (Sm/Co = 1:2) NPs + NIR + M group shows the most significant inhibitory effect on tumor progression, illustrating a satisfactory therapeutic effect.Comparatively, the 4T1 tumor-bearing mice still survive after 60 days of treatment in the mSiO 2 -SmCo x (Sm/Co = 1:2) NPs + NIR + M group with markedly prolonged lifetime, confirming again the superior anticancer efficacy (Figure 6B).As displayed in Figure 6E, the infrared thermal images of the tumor site were acquired at different times, where the skin temperature of the 3) and 4) groups increased notably, demonstrating the excellent magnetic and photothermal effect of mSiO 2 -SmCo x (Sm/Co = 1:2) NPs.The hematoxylin and eosin (H&E) staining photographs of the prepared tumor section show that the most obvious apoptosis of tumor cells occurs in the mSiO 2 -SmCo x (Sm/Co = 1:2) NPs + NIR + M group (Figure 6F).

Conclusion
In summary, we demonstrated the superior magnetic/ photothermal effect-induced hyperthermia therapy of mSiO 2 -SmCo x (Sm/Co = 1:2) NPs to regard as an advanced synergistic hyperthermia therapeutic paradigm.The well-dispersive mSiO 2 -SmCo x NPs were rationally constructed through a one-step procedure among the regulated theoretical molar ratio of Sm/ Co among 1:1, 1:2, and 1:4 for controlling the dispersion and magnetism properties of SmCo x NPs in situ growth in the pore structure of mSiO 2 .The diverse porous structures and high specific surface of mSiO 2 areas were utilized for locating the permanent magnetic SmCo x NPs.The mSiO 2 -SmCo x (Sm/Co = 1:2) NPs with highly dispersed morphology have an average diameter of ~73.08 nm and the photothermal conversion efficiency was determined to be nearly 41%.The in vitro and in vivo anti-tumor evaluation of mSiO 2 -SmCo x (Sm/Co = 1:2) NPs demonstrated the promising potential for hyperthermiainduced tumor therapy due to magnetic and photothermal effects.

FIGURE 6 (
FIGURE 6 (A) Biodistribution of Sm ions (% injected dose (ID) of Sm per Gram of tissues) in main tissues and tumor in 1, 3, 6, 12, and 24 h of intravenous administrations of mSiO 2 -SmCo x (Sm/Co = 1:2) NPs (n = 3).(B) Changes in the average body weight, (C) relative tumor volume, and (D) survival rates of mice with various treatments.(E) Temperature elevation at the tumor sites of 4T1 tumor-bearing mice under 808 nm laser (1.0 W cm -2 ) irradiation and 808 nm laser irradiation in magnetic conditions with mSiO 2 -SmCo x (Sm/Co = 1:2) NPs for 4 min (F) H&E-stained photographs of tumor slices obtained from tumor-bearing mice after treatments.Error bars are based on the standard errors of the mean.Statistical analysis is assessed by unpaired Student's two-sided t-test.***p < 0.001, **p < 0.01, or *p < 0.05.