Achieving enhanced NIR light-induced toxicity via novel hybrid magnetic nanoparticles

Abstract

Magnetic nanomaterials have been widely used in biomedical fields due to their non-toxicity and versatile functionalities. In this study, we rationally synthesized novel Zn²⁺-doped magnetic nanoparticles via a facile but organic reagent-free hydrothermal route, as well as utilized them to achieve enhanced NIR light-induced toxicity towards cancer cells. By using other metal salts as precursors in the typical synthesis, a series of hybrid magnetic nanoparticles could be obtained with inexpensive inhesion. Upon an 808 nm laser irradiation, Zn²⁺-doped magnetic nanoparticles revealed excellent photothermal effect with high photo-stability and a concentration/time-dependent manner. Cytotoxicity studies of these nanoparticles suggested their low systemic toxicity towards 786-O cells. After efficient endocytosis, Zn²⁺-doped magnetic nanoparticles were mainly accumulated in lysosomes, which caused the persistent release of Zn²⁺ ions and the following generation of reactive oxygen species (ROS). Significantly, enhanced generation of ROS was only detected in the group treated with magnetic nanoparticles and a NIR light irradiation because the localized heat could increase the dissolution of magnetic nanoparticles in the acid medium. The combination of toxic ROS and photothermal effect could minimize the dosage of nanoagents and enhance the NIR light-induced toxicity. More importantly, Zn²⁺-doped magnetic nanoparticles showed more toxicity than pure Fe₃O₄ nanoparticles prepared with a similar route upon the irradiation. Last but not least, our study demonstrated a new concept by using single-phase hybrid magnetic nanoparticles as promising candidates for NIR light-induced cancer treatment and brought more meaningful ideas in the bio-related research.

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Introduction

Cancer has been recognized as a leading cause of death worldwide for centuries according to the World Cancer Report. To efficiently ablate tumours, various therapies including surgery, chemotherapy, and radiotherapy have been developed and extensively employed. Although promising, these approaches often suffer from the probability of cancerous metastasis, the risk of injuring normal organs/tissues, and the toxic effect towards immune system.¹ Recently, near-infrared (NIR) laser-mediated strategies including photothermal therapy (PTT) and photodynamic therapy (PDT) have turned into significant routes for cancer treatments both in lab and in clinic.^2–4 By using NIR light as an external stimulus for spatial/temporal exposure, these phototherapies can maximize the therapeutic efficacy and minimize the side effects. During the PTT/PDT protocol, heat and reactive oxygen species (ROS) are highly required to generate around the tumorous regions for killing cancer cells. More importantly, singlet O₂-enabled treatment and localized “burning” hyperthermia also provide additional advantages including facile procedure, fewer complications, and shorter period in hospital.^5–7

To data, the use of novel nanomaterials as phototherapeutic agents against tumours has been widely explored.^8–12 For instance, carbon-based nanomaterials, metallic nanoparticles, and organic nanoparticles have exhibited their excellent photothermal effect in cancer treatment.^13–16 Multifunctional up-conversion nanocomposites have been utilized to convert 980 nm NIR light to visible light, which can act as the excitation of photosensitizers to exert PDT effect.^17–20 Moreover, recent studies have demonstrated that CuS nanoparticles, PEGylated W₁₈O₄₉ nanomaterials, and multi-branched gold nanoechinus with dual modal photothermal/photodynamic effects show their talent completely in biomedicine.^21–26 However, some intrinsic limitations of these nanoagents may confined their usages. For example, noble metallic nanoparticles with unclear long-term toxicity cannot be metabolized from bodies.^27–29 Serious pulmonary inflammation and oxidative stress can be found after intravenous administration of carbon nanotubes.^30–32 The restricted choice of excitation wavelengths and related low quantum yields of up-conversion nanomaterials limits their translation from lab to clinic.^33–36 Considering these unwanted shortcomings, it is highly desired to design and develop novel phototherapeutic agents with high bio-compatibility which are able to benefit the daily demand in clinic.

Magnetic nanoparticles have gained tremendous attention owing to their non-toxicity, high bio-compatibility, and unique magnetic properties.^37–40 Current research based on magnetic nanomaterials has been extensively performed on biomolecule separation, targeted drug/gene delivery, magnetic resonance imaging (MRI), and hyperthermia treatment.^41–44 Significantly, superparamagnetic nanoparticles have been approved as T₂-weighted MRI contrast agents by the Food and Drug Administration. Otherwise, localized magnetic fluid-based hyperthermia upon an alternating magnetic field (AMF) has been widely used recently. Thus, magnetic nanoparticles are quite suitable for bio-related usages because of their acknowledged advantages. Several studies indicate that NIR light-induced photothermal effect of magnetic nanoparticles can efficiently kill cancer cells and bacteria.^45–49 However, the high-dose magnetic nanoparticles used in the photothermal treatment may cause potential toxicity and seriously limit their usages. Accordingly, phototherapeutic system based on magnetic nanoparticles must be improved.

Currently, hybrid magnetic nanoparticles have been used in AMF-induced hyperthermia and medical imaging.^50–52 However, to the best of our knowledge, the use of single-phase hybrid magnetic particles as high-performance phototherapeutic agents has not been mentioned till now. Herein, we reported the preparation of novel Zn²⁺-doped magnetic nanoparticles and utilize them to achieve enhanced NIR light-induced toxicity towards cancer cells. By changing the metal precursors, a series of hybrid magnetic nanoparticles were obtained via a similar route. As expected, Zn²⁺-doped magnetic nanoparticles showed great photothermal effect with high photo-stability upon the irradiation of an 808 nm laser. More importantly, Zn²⁺-doped magnetic nanoparticles could lead to more toxicity than pure Fe₃O₄ nanoparticles upon the same irradiation condition. We ascribed this enhanced anticancer activity to the increased release of Zn²⁺ ions in the acid condition and the following generation of ROS from these nanoparticles. Thus, the systemic combination of photothermal effect and toxic ROS showed its full advantages in biomedicine.

Experimental details

Chemicals

Fluorescein isothiocyanate (FITC), polyethylene glycol-2000, calcein AM, propidium iodide (PI), urea, ZnCl₂, MnCl₂·4H₂O, MgCl₂·6H₂O, NiCl₂·6H₂O, FeCl₃·6H₂O, and sodium citrate were obtained from Sigma-Aldrich. All chemical agents were of analytical grade and used directly without any purification. Deionised (D.I.) water was used all through the experiments.

Synthesis of Zn²⁺-doped magnetic nanoparticles

Zn²⁺-Doped magnetic nanoparticles were prepared via a facile hydrothermal method. Typically, PEG-2000 (0.4 g), urea (6 mmol), ZnCl₂ (0.5 mmol), FeCl₃·6H₂O (1 mmol), and sodium citrate (3 mmol) were dispersed in D.I. water (40 mL) under magnetic stirring. 1 h later, above mixture was transferred to a Teflon-lined stainless steel autoclave (50 mL), which was heated at 200 °C. 12 h later, the autoclave was cooled to room temperature naturally. The resulting Zn²⁺-doped magnetic nanoparticles were obtained via magnetic separation, washed with ethanol, and dried in vacuum.

Synthesis of other metal-doped magnetic nanoparticles

To achieve other hybrid magnetic nanoparticles, MgCl₂·6H₂O (0.5 mmol), MnCl₂·4H₂O (0.5 mmol), and NiCl₂·6H₂O (0.5 mmol) were used as metal precursors instead of ZnCl₂.

Synthesis of Fe₃O₄ nanoparticles without any doping

To obtain pure Fe₃O₄ nanoparticles, FeCl₃·6H₂O (2 mmol) was used as metal precursor instead of ZnCl₂ and FeCl₃·6H₂O.

Modification of Zn²⁺-doped magnetic nanoparticles with FITC

Zn²⁺-Doped magnetic nanoparticles (100 mg) and FITC (0.5 mg) were dispersed in anhydrous tetrahydrofuran (THF, 30 mL) by ultrasonication. This solution was then subject to mechanical stirring for another 2 h. With the assistance of a magnet, FITC-modified Zn²⁺-doped magnetic nanoparticles were separated, washed with ethanol, and dried in vacuum for further usage.⁵³

Cell culture

786-O cells (human kidney carcinoma cell line) and 4T1 cells (mouse breast cancer cell line) were supplied by American Type Culture Collection (ATCC). 786-O cells and 4T1 cells were cultured in a 1640 medium and DMEM, respectively. Both mediums contained 10% fetal bovine serum, penicillin (100 U mL⁻¹), and streptomycin (100 U mL⁻¹). Cells were incubated in a humidified incubator at 37 °C and 5% CO₂, harvested with trypsin, and re-suspended in a fresh medium before usage.

Time-dependent cellular internalization

FITC-modified Zn²⁺-doped magnetic nanoparticles were used to explore the endocytosis process. 786-O cells were cultured in a 6-well plate to allow the attachment. 12 h later, FITC-modified Zn²⁺-doped magnetic nanoparticles (200 μg mL⁻¹) were added into culture medium. At any expected time, cells were washed with 0.9% NaCl solution, stained with LysoTracker Red, and observed under a fluorescence microscope.

Cytotoxicity

MTT assay was carried out to quantify the cytotoxicity of Zn²⁺-doped magnetic nanoparticles. Typically, 786-O cells or 4T1 cells were cultured in 96-well plates with a density of 5 × 10³ per well. 12 h later, Zn²⁺-doped magnetic nanoparticles with different concentration were added into above culture medium. 24 h after incubation with these nanoparticles, MTT reagent was added into each well. 4 h later, supernatant was removed and formazan crystals were dissolved by DMSO. The absorbance at 490 nm was measured via a microplate reader.

Detection of ROS level

786-O cells with a density of 1 × 10⁴ were cultured in a 6-well plate for 12 h. Cells were then incubated with Zn²⁺-doped magnetic nanoparticles (400 μg mL⁻¹) for 4 h and irradiated with a 808 nm laser (2 W cm⁻², 5 min). Above cell medium was replaced with DCFH-DA solution and incubated for another 0.5 h at 37 °C in dark. The groups without any treatment and treated with only NIR laser irradiation and nanoparticles were named as control, NIR, and NPs. Images were collected on a fluorescence microscope.

Quantitative and visible photothermal cytotoxicity

To quantitatively investigate the NIR light-induced cytotoxicity of Zn²⁺-doped magnetic nanoparticles, 786-O cells or 4T1 cells were incubated in 96-well plates with a density of 5 × 10³ per well at 37 °C for 12 h. Zn²⁺-doped magnetic nanoparticles or pure Fe₃O₄ nanoparticles with different concentrations were added to the culture medium. 3 h later, cells were exposed to an 808 nm laser with an irradiation intensity of 2 W cm⁻². Then, all the cells were incubated for another 24 h. Cellular viability were evaluated via MTT assay. To obtain visible results, 786-O cells and 4T1 cells with a density of 5 × 10⁴ were incubated in 6-well plates for 12 h. Zn²⁺-doped magnetic nanoparticles (400 μg mL⁻¹) were added to the medium. Cells were irradiated via an 808 nm laser with a power density of 2 W cm⁻² for 5 min. Cells were stained with calcein AM and PI. Dual-stained images were collected on a fluorescence microscope.

Statistical analysis

All data were expressed as the mean result ± standard deviation (SD). Statistical analysis was performed via Origin 8.0 software.

Result and discussion

Fig. 1 illustrated the rational synthesis of various hybrid magnetic nanoparticles via a facile hydrothermal route, and the usage of Zn²⁺-doped ones as enhanced NIR light-associated phototherapeutic agents against cancer cells via photothermal effect and ROS injury. Compared with other synthetic methods, our strategy held many advantages. First, our hydrothermal synthesis occurred in aqueous solution without the application of any organic solvent and all the reagents were less expensive. Second, by changing the metal precursors, a series of hybrid magnetic nanoparticles could be achieved. Third, these hybrid magnetic nanoparticles showed large saturation magnetization and excellent water solubility, which was highly important in biomedical applications.

Fig. 1 Schematic illustration of the one-pot hydrothermal synthesis of various hybrid magnetic nanoparticles (A) and the usage in enhanced NIR light-associated cancer therapy via Zn²⁺-doped magnetic nanoparticles (B).

Morphological and structural features of typical Zn²⁺-doped magnetic nanoparticles were examined via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM image and TEM image revealed that these nanoparticles showed a uniform and non-aggregated nature with an average diameter of 150 nm and a related coarse surface (Fig. 2A and B). High-resolution TEM image indicated that these nanoparticles were composed of many smaller nanocrystals. Wide-angle XRD pattern revealed a cubic ferrite spinel phase of this product (Fig. 2C). The interplanar distance could be calculated as 0.48 nm, which was highly consistent with the separation between the (111) lattice planes. Moreover, these Zn²⁺-doped magnetic nanoparticles held a diffraction-rings characteristic of polycrystalline material based on the selective area electronic diffraction pattern. As shown in Fig. 2D, room-temperature magnetic measuration indicated that these Zn²⁺-doped magnetic nanoparticles held a saturation magnetization value of 76.1 emu g⁻¹ with a superparamagnetic characteristic.

Fig. 2 SEM image (A), TEM image (B), wide-angle XRD pattern (C), and magnetic hysteresis loop (D) of Zn²⁺-doped magnetic nanoparticles. Inset of (B) HR-TEM image. Inset of (C) SAED image.

FT-IR spectra further confirmed the successful coating of PEG and citrate molecules on the surface of Zn²⁺-doped magnetic nanoparticles via the presence of carboxylate vibrations and C–H vibrations (Fig. S1A†). Based on the thermogravimetric analysis, the amount of these functionalized groups could be calculated as 13.85% (Fig. S2B†). Identified by ICP-MS method, elemental composition of these nanoparticles could be defined as Zn_0.4Fe_0.6Fe₂O₄. Similar with previous studies, both PEG molecules and citrate molecules played important roles in the typical synthesis.^53–55 Similar with a previous study, PEG molecules could enhance the monodispersity of Zn²⁺-doped magnetic nanoparticles and decrease their aggregation during the synthesis (Fig. S2†).⁴⁴ Without the addition of PEG molecules in the typical synthesis, these Zn²⁺-doped ones exhibited serious aggregation based on the SEM image. Citrate molecules could lead to a slow crystallization via the efficient coordination with Zn²⁺ ions and act as reducing reagents under the high-temperature condition.

To benefit the understanding of readers, time-dependent experiments were designed to explore the formation of Zn²⁺-doped magnetic nanoparticles (Fig. 3). Time-dependent SEM images indicated a step-by-step nucleation process. In detail, no product could be detected when the initial solution was treated for 1 h. A little amorphous product could be harvested when the period was 3 h. We could find characteristic peaks of spinel ferrite in XRD pattern by extending the period to 6 h. When the period was prolonged to 12 h, the product could be totally transformed into well-crystallized nanoparticles. By using different metal precursors, our resent synthesis was also adapted to the synthesis of other magnetic nanoparticles. For instance, Mg²⁺-, Mn²⁺-, and Ni²⁺-doped magnetic nanoparticles were obtained via a similar route. Fig. 4, S3, and Table S1† provided the detailed information of these hybrid magnetic nanoparticles. Elemental compositions of these nanoparticles could be calculated as Mg_0.4Fe_0.6Fe₂O₄, Mn_0.7Fe_0.3Fe₂O₄, and Ni_0.3Fe_0.7Fe₂O₄ via ICP-MS analysis. All these products revealed high crystallizability and excellent magnetic property.

Fig. 3 SEM images of Zn²⁺-doped magnetic nanoparticles obtained at 3 h (A), 6 h (B), 9 h (C), and 12 h (D). Time-dependent wide-angle XRD patterns of above samples (E).

Fig. 4 SEM images of Mg²⁺- (A), Mn²⁺- (B), and Ni²⁺-doped magnetic nanoparticles (C). Wide-angle XRD patterns of above samples (D).

Fig. 5A illustrated the UV-vis-NIR spectra of Zn²⁺-doped magnetic nanoparticles with different concentrations. These as-synthesized nanoparticles with a deep black colour exhibited a broad absorption band ranging from UV to NIR wavelengths. As expected, the value of absorbance highly enhanced with the increasing of the agent concentrations. To investigate the photothermal effect, Zn²⁺-doped magnetic nanoparticles were dispersed in 0.9% NaCl solution with different concentrations, and irradiated with an 808 nm NIR laser. In our design, 0.9% NaCl solution was selected as a negative control. All the temperatures of samples containing nanoparticles followed a concentration/irradiation period-dependent manner (Fig. 5B). Samples containing nanoparticles with higher concentrations performed better than those with lower concentrations. Otherwise, the longer the irradiation period, the higher the sample temperature was. For example, the temperature raised by Zn²⁺-doped magnetic nanoparticles could reach 17.5 °C with a concentration of 0.8 mg mL⁻¹ and a NIR exposure period of 10 min. However, the temperature of 0.9% NaCl solution was only increased by 3.9 °C upon the same treatment. Previous studies demonstrated that cells could be killed after treatment at 42 °C for half an hour, while this treatment could be shortened to less than 10 min for temperature over 45 °C. According to our present data, cells could be easily heated to 45 °C within 10 min and be killed upon the incubation of hybrid magnetic nanoparticles and a NIR laser irradiation. In addition, the photostability of these hybrid magnetic nanoparticles were evaluated. TEM images revealed that no differences happened in morphology and size of these nanoparticles before and after a long-term irradiation with a NIR laser (Fig. 2B and S4†). These results further implied that our nanoparticles exhibited high photostability and held more potential in biomedicine.

Fig. 5 UV-vis spectra of Zn²⁺-doped magnetic nanoparticles with different concentrations (A). Photothermal effect of 0.9% NaCl solution containing Zn²⁺-doped magnetic nanoparticles treated with a NIR light with an irradiation intensity of 2 W cm⁻² (B).

Prior to study the phototherapeutic effect on cancer cells of these Zn²⁺-doped magnetic nanoparticles upon a NIR laser irradiation, MTT assay and calcein AM/PI dual-stained imaging associated with 786-O cells were explored at first. As shown in Fig. S5,† all the viabilities of 786 cells were not hindered by these nanoparticles even upon the maximal co-incubation concentration of 1 mg mL⁻¹. To obtain more accurate results, dual-stained fluorescence images were collected to confirm the visualized cellular viability in the presence of Zn²⁺-doped magnetic nanoparticles. As shown in Fig. S6,† we could not detect red fluorescence from PI-stained dead cells in the group treated with nanoparticles. As compared to the control group, there were no obvious differences occurred in the morphology of 786-O cells treated with nanoparticles, indicating that 786-O cells were still alive. Moreover, cellular morphology in the groups treated with nanoparticles was found to be as normal as those in the control group and exhibited a flattened appearance. On the basis of these results, our well-synthesized Zn²⁺-doped magnetic nanoparticles held high bio-compatibility.

Previous studies demonstrated that magnetic nanoparticles based on iron oxide could be dissolved in acidic medium and release metal ions. We then investigated that whether zinc ions could be released from our nanoparticles in an acidic condition. Treated with an acidic buffer, a clear pH-dependent ionization of these nanoparticles and persistent release of Zn²⁺ ions were observed (Fig. 6A). However, we could not find the release of Zn²⁺ ions at neutral pH from these nanoparticles. Accordingly, these results indicated that Zn²⁺-doped magnetic nanoparticles could be dissolved in an acidic condition. Then we used FITC-modified Zn²⁺-doped magnetic nanoparticles to explore their cellular uptake process. These fluorescent hybrid magnetic nanoparticles were prepared via a previous route.⁵³ In THF, chemical reaction between isocyanate group in FITC and carboxyl group in magnetic nanoparticles occurred, which leaded to the formation of acid amide and carbon dioxide. The formation of acid amide could effectively link fluorescence FITC with magnetic nanoparticles. As a routine lysosome label, lyso-tracker red was used to spatially confirm the precise position of these nanoparticles. Time-dependent cellular uptake of FITC-modified nanoparticles was illustrated in Fig. 6B. Compared with the group treated with 3 h incubation, we could detect more green fluorescence of FITC in the group treated with 6 h incubation, which indicated that more FITC-modified Zn²⁺-doped magnetic nanoparticles could be uptaken by 786-O cells along with the time passing. Owing to the acidic environment of lysosome, Zn²⁺ ions could be released from these nanoparticles after cellular uptake, which could cause further generation of ROS.

Fig. 6 Time-dependent ionization of Zn²⁺-doped magnetic nanoparticles in different buffer systems (A). Fluorescence and optical images of 786-O cells incubated with FITC-modified Zn²⁺-doped magnetic nanoparticles (B). The scale bar is 20 μm.

After understanding the efficient cellular uptake of Zn²⁺-doped magnetic nanoparticles, intracellular NIR light-induced ROS generation was then explored. To obtain precise evidence, intracellular ROS evaluation was assessed via fluorescence microscopy. In detail, ROS generation was confirmed by detecting the green fluorescence of 2,7-dichlorofluorescein (DCF), which developed from ROS-treated non-fluorescence dichlorofluorescein diacetate (DCFH-DA). Green fluorescence with higher intensity could be found in the NPs + NIR group (Fig. 7). Moreover, quantitative data of flow cytometry further confirmed our above results that there was the most ROS generation in the group of NPs + NIR (Fig. S7†). However, nearly no green fluorescence could be detected in the control group, the NPs group, and the NIR group, which demonstrated that the present NIR laser irradiation could not enhance the generation of ROS. Generally, ROS could induce serious cellular death or injury. Thus, more cellular death might occur in the group of NPs + NIR, which provided more opportunities for Zn²⁺-doped magnetic nanoparticles as high-performance phototherapeutic agent with enhanced light-induced toxicity.

Fig. 7 Levels of ROS generation in 786-O cells via fluorescence analysis after various treatments. The scale bar is 100 μm.

To explore the NIR light-induced toxicity towards cancer cells of Zn²⁺-doped magnetic nanoparticles, MTT assay was used to determine the viabilities of 786-O cells. As shown in Fig. 8A, our nanoparticles leaded to a drastic drop in all the percentages of cellular viabilities after a NIR light irradiation. The killing efficacy held a concentration/irradiation period-dependent manner. Different phototherapeutic effects thereby could be obtained by regulating the irradiation periods of NIR light and incubated concentrations of nanoparticles. These results implied that Zn²⁺-doped magnetic nanoparticles were active after the irradiation with a NIR light. To further obtain visible results of cellular viability, calcein AM/PI dual-stained images were collected on a fluorescence microscopy. No differences were found between the control group, the NIR group, and the NPs group (Fig. 8B). All the 786-O cells in above three groups were alive, indicating the high bio-compatibility of these hybrid magnetic nanoparticles and negligible photo-induced toxicity of our present irradiation condition. However, nearly all the cancer cells were killed in the NPs + NIR group, which was highly consistent with our MTT assay. We ascribed this cellular death in the NPs + NIR group to the systemic cooperation of photothermal effect and ROS injury during the phototherapeutic process. Upon a NIR light irradiation, the photothermal effect of these nanoparticles could enhance the release of Zn²⁺ ions and the generation of ROS with a high speed. However, without a NIR light irradiation and related photothermal effect, the generation of ROS in the NPs group was quite slow. Along with the generation of ROS, intracellular enzymes could clear up them via intrinsic self-repair effect step by step in the NPs group, which thus could not cause serious cellular injury. To explore the extensive usages of Zn²⁺-doped magnetic nanoparticles, 4T1 cells were selected to confirm our above results. All the cellular viabilities were not inhibited by these nanoparticles. Moreover, present irradiation condition did not cause cellular death. Serious cellular injury only occurred in the group of NPs + NIR and followed a concentration/irradiation period-dependent manner (Fig. S8†).

Fig. 8 Viabilities of 786-O cells incubated with Zn²⁺-doped magnetic nanoparticles treated with a NIR light irradiation (A). Fluorescence images of calcein AM/PI-stained cells after various treatments (B). The scale bar is 200 μm.

To investigate the detailed role of Zn²⁺ ions in our study, pure Fe₃O₄ nanoparticles were synthesized via a similar route (Fig. S9†). SEM image and TEM image indicated that pure Fe₃O₄ nanoparticles with an average diameter of 140 nm held a sphere character. Wide-angle XRD pattern indicated their cubic ferrite spinel structure. EDS analysis showed that these pure Fe₃O₄ nanoparticles were made up of Fe, C, and O. The saturation magnetization of pure Fe₃O₄ nanoparticles could be calculated as 76.8 emu g⁻¹, which was much higher than Mg²⁺-, Mn²⁺-, and Ni²⁺-doped ones (Table S1†). However, Zn²⁺-doped ones held a similar saturation magnetization value (76.1 emu g⁻¹) with pure Fe₃O₄ nanoparticles, which could be attributed to the doping amount of metal ions in magnetic nanoparticles and intrinsic electronic/magnetic properties of doping metal ions.^53–55 UV-vis spectra of pure Fe₃O₄ nanoparticles revealed that nearly no differences were detected between pure ones and Zn²⁺-doped ones in absorption values ranging from UV region to visible region. These exciting results thus indicated that pure Fe₃O₄ nanoparticles might hold similar photothermal effect with Zn²⁺-doped ones in tube experiments. Detailed phototherapeutic effects of pure Fe₃O₄ nanoparticles towards both 786-O cells and 4T1 cells were provided in Fig. S10.† Significantly, the NIR light-induced toxicity by using pure Fe₃O₄ nanoparticles as agents was much lower than those treated with Zn²⁺-doped ones assistanced with the same irradiation condition. These results demonstrated that Zn²⁺ ions in our hybrid nanoparticles played an important role in killing cancer cells. During the phototherapeutic process, photothermal effect of our nanoparticles could enhance the release of Zn²⁺ from these hybrid magnetic nanoparticles and increase the generation of ROS. These ROS together with the excellent photothermal effect thus could cause serious cellular injury and death. Thus, Zn²⁺-doped magnetic nanoparticles could act as efficient phototherapeutic agents to obtain enhanced NIR light-induced toxicity towards cancer cells as compared to pure Fe₃O₄ nanoparticles. Previous study demonstrated that a series of hybrid magnetic nanoparticles with different doping were achieved by changing the reaction conditions.⁵⁵ Therefore, our present route could be extended to the preparation of various hybrid magnetic nanoparticles with different doping levels. More importantly, these Zn²⁺-doped magnetic nanoparticles with different doping levels might hold different anticancer activity upon the irradiation of NIR light both in vitro and in vivo. Further discussion and experiments thus were highly needed and still in their progress.

Conclusions

In summary, we have rationally designed and synthesized novel Zn²⁺-doped magnetic nanoparticles via an economical hydrothermal route, and utilized them to achieve enhanced NIR light-induced toxicity towards cancer cells in vitro. Detailed studies about the time-dependent formation process of these nanoparticles were discussed. Otherwise, a series of hybrid magnetic nanoparticles were successfully achieved by using Mg²⁺, Mn²⁺, and Ni²⁺ salts as metal precursors in the typical synthesis. Cytotoxicity suggested that Zn²⁺-doped magnetic nanoparticles showed negative effects on the morphology and viability of 786-O cells. After efficient endocytosis, Zn²⁺-doped magnetic nanoparticles were mainly accumulated in lysosomes, which caused the release of Zn²⁺ and further generation of ROS. Significantly, enhanced generation of ROS could be detected in the NPs + NIR group. Accordingly, toxic ROS together with excellent photothermal effect of these Zn²⁺-doped magnetic nanoparticles upon a NIR light irradiation could minimize the dosage of photothermal agents and enhance the light-induced toxicity towards cancer cells. More importantly, Zn²⁺-doped magnetic nanoparticles showed more toxicity than pure Fe₃O₄ nanoparticles upon the same irradiation condition. Last but not least, this study provided a new concept by using hybrid magnetic nanoparticles as promising candidates for NIR light-induced cancer treatment and revealed great potential for further biomedical usages.

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Footnote

† Electronic supplementary information (ESI) available: Supporting figures and table. See DOI: 10.1039/c6ra10513k

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