A Dual-Functional Ferroferric Oxide/Quantum Dots Theranostic Nanoplatform for Fluorescent Labeling and Photothermal Therapy

silica structure on the nanoplatform, lowering the mass of the nanoplatform and effectively promoting the absorption efficiency of the incident light compared with the traditional silica layer. In addition, after endocytosis of the nanoplatform, cancer cells are easily detected under a fluorescence microscope because of the excellent fluorescent behavior of QDs. Moreover, in vitro experiments confirm that nanoplatform possesses perfect photothermal effect to destroy tumor cells under laser irradiation. Therefore, ferroferric oxide/QDs nanoplatforms, combined with the functions of fluorescent labeling and photothermal therapy for cancer cells, are expected to be a promising biopotential material in the field of diagnosis and treatment. for 2 h at 37 ° C with 5% CO 2 (v/v). Finally, the glass coverslip was washed with PBS solution for three times to remove the unuptaked nanoplatforms, and then the cancer cells were prepared to detect the labeling behavior under fluorescence microscopy. Photothermal Therapy In Vitro : On the one hand, aqueous solutions of Fe 3 O 4 /QDs nanoplatform samples were irradiated by an 808 nm laser with different concentration. The temperature of all samples was recorded, and all the dates were obtained from the average of five times measurement. On the other hand, the photothermal therapy for cancer cells was investigated. As harvested from the culture plate and coincubation with adequate nanoplatform, the cancer cells were washed with excess PBS solution. Then the cancer cells were irradiated by an 808 nm laser with the power density of 1.0 W cm − 2 . After irradiated for 10 min, the cancer cells were stained to examine the live and dead cells under fluorescence microscopy. Characterization : The crystal structure was measured by X-ray diffractometer (Rigaku-Dmax 2500) with Cu K α (0.15 405 nm) radiation. The morphologies were inspected on TEM (JEM-1400) with acceleration voltage of 200 kV. Magnetic measurement was performed by vibrating sample magnetometer (Quantum Design) at 300K. The photoluminescence (PL) emission spectra were examined by spectrofluorimeter (Hitachi F-4500) equipped with Xenon lamp as the light source.

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/ppsc.202100043.

DOI: 10.1002/ppsc.202100043
(Fe 3 O 4 ) nanoparticles, possessing the advantages of easy-synthesis process and environmental friendliness, were pursued and analyzed by biomedical scientists in the recent years. [9][10][11] Photothermal effect, as a typical and outstanding property of Fe 3 O 4 nanoparticles, was a photothermal conversion process, during which the irradiation light energy was absorbed by the nanoparticle and transformed into local hyperthermia. [12][13][14] As the nanoparticle was transported to targeted cancer cells, the local hyperthermia would generate high temperature and lead to the cancer cell inactivation. Gupta et al. reported that eugenate capped Fe 3 O 4 nanoparticles exhibited a distinct light absorption behavior and were successfully employed to destroy the deep tissue based on the photothermal therapy. [15] Besides, the Fe 3 O 4 nanoparticle with a size of smaller than 20 nm exhibits excellent superparamagnetic behavior. Under high gradient external magnetic field, drug molecules banded onto the biocompatible Fe 3 O 4 nanoparticles could be easily delivered to the targeted region. [16] Coprecipitation method and organometallic decomposition method were the main approaches to obtain high-quality Fe 3 O 4 nanoparticles. [17,18] Though the organometallic decomposition method played a significant role to prepare superior Fe 3 O 4 nanoparticle with perfect crystal texture and high monodispersity, the hydrophobic property of the obtained nanoparticles seriously restricted the biomedical application in vivo experiment. Due to the intrinsic magnetism, the Fe 3 O 4 nanoparticles tend to attract each other and produce the phenomenon of agglomeration, leading to the occurrence of precipitation. Therefore, a further decoration on the Fe 3 O 4 nanoparticles was a necessary step to ensure them good biocompatibility and stable circulation in the biomedical applications. In the previous reports, [19][20][21] silica (SiO 2 ), as a suitable inter-carrier, not only could be easily modified onto the surface of Fe 3 O 4 nanoparticles to avoid the appearance of precipitation, but also could be functioned with different groups to connect with other molecules. Besides, it was well-known that the irradiation light could be strongly absorbed or scattered by encountered medium, resulting in the reduction of irradiation light intensity. A mesoporous structure of silica was a suitable substitution to lower the density of silica and enhance the transmissivity of irradiation light, avoiding the side effect of absorption and scattering. [22,23]

Introduction
With the rapid development of science and technology, nanomedical materials had gain extensive attention and been applied in the field of medical diagnosis and treatment, such as photothermal therapy, [1,2] chemodynamic therapy, [3][4][5] fluorescent labeling, [6,7] and magnetic resonance imaging. [8] Ferroferric oxide www.advancedsciencenews.com www.particle-journal.com In the field of disease diagnosis, positron emission tomography, magnetic resonance imaging, and optical imaging were the primary imaging techniques for noninvasive detection in vivo. [24,25] Among them, great interest had been devoted to the optical imaging technology due to its high resolution, easy operation, and low cost. Superior to the organic fluorescent dye, quantum dots (QDs) possessed many promising physicochemical properties, including size-dependent emission bands, superior photostability, and larger Stokes shifts. [26][27][28] However, the problem of the intrinsic toxicity of traditional Cd-based QDs seriously limited their further clinical use. In order to solve the toxicity restriction, new type nontoxic Mn-doped ZnS QDs, consisting of Mn ion as the emission center and ZnS as the host, were introduced by scientists in the past few years. Another advantage about the Mn-doped ZnS QDs was that the emission behavior was not seriously affected by the relatively poor crystal texture. Recently, our group reported a Mn-doped ZnCdS/ZnS core/shell QDs, possessing the advantages of tunable excitation wavelength and bright fluorescent behavior, were successfully employed to label the targeted cancer cell.
Though individual Fe 3 O 4 nanoparticle and QDs exhibited excellent photothermal effect and fluorescent behavior respectively, there was still much space to engineer a new complex nanostructure by integrating the Fe 3 O 4 nanoparticle and QDs. Here, we designed a theranostic nanoplatform, decorating Mn-doped ZnS/ZnS QDs onto the surface of mesoporous SiO 2 coated Fe 3 O 4 nanoparticle based on the electrostatic interaction for the first time. During the coating progress of SiO 2 onto Fe 3 O 4 nanoparticle, larger pores on the internal SiO 2 layer were formed relying on synergistic effect of hexadecyltrimethyl ammonium bromide and pore-enlarging agent. The as-synthesized nanoplatform exhibited superparamagnetism, and could easily be dragged to the direction of the external magnet. In addition, the biological experiments demonstrated that the endocytosis of the nanoplatform by cancer cells was efficient, which could be confirmed by the successful detection of the cancer cells under fluorescence microscope. Furthermore, after irradiation exposure, irreversible destruction to the cancer cells was readily examined. In conclusion, the nanoplatform realized simultaneous magnetic separation, fluorescence imaging, and photothermal therapy. We believed that such a compositive nanoplatform had the potential to successfully challenge some of the currently limitations in medical diagnosis and treatment.

Results and Discussion
Recently, great effect had been devoted to synthesize complex nanostructures to satisfy the need of multiple functions in the biomedical research. [29,30] However, the disadvantage of biological toxicity severely limited the clinical application. Here, a layer-by-layer assembly was employed to design a biocompatible, nontoxic Fe 3 O 4 /Mn-doped ZnS/ZnS nanoplatform, which was suitable for photothermal therapy and targeted labeling for cancer cells. As described in Figure 1, the nanoplatform was prepared using Fe 3 O 4 core for the photothermal therapy and effective magnetic separation, Mn-doped ZnS/ZnS QDs for targeted labeling, mesoporous SiO 2 for structural stability, and folic acid for good endocytosis. Due to the loose structure of mesoporous SiO 2 , the mass density of the nanoplatform was effectively lowered, and much radiation light could be permeated and absorbed by the Fe 3 O 4 core, exhibiting a much higher efficiency of photothermal therapy for cancer cells.
Gram-scale hydrophilic Fe 3 O 4 nanoparticles were fabricated by a facile coprecipitation approach using ammonia as catalyst and oleic acid as surface stabilizer. [31] As shown in Figure 2a, the hydrosoluble Fe 3 O 4 nanoparticles had a uniform size distribution of around 11 nm. We introduced the oleic acid molecule as surface stabilizer, thus producing the excellent dispersibility. However, it was well-known that the Fe 3 O 4 nanoparticles tend to attract each other and formed a larger cluster originating from the intrinsic anisotropic dipolar attraction, leading to the  www.advancedsciencenews.com www.particle-journal.com loss of superparamagnetism. A silica layer was grown onto the surface of Fe 3 O 4 nanoparticle under vigorous stirring, in order to protect the Fe 3 O 4 nanoparticle from aggregation and keep them in a stable status. In addition, surfactant-mediated templating method was introduced to produce sufficient pores with larger volume on the silica layer using hexadecyltrimethyl ammonium bromide (CTAB) as template, and 1, 3, 5-trimethylbenzene (TMB) as pore swelling agents. Figure 2b showed the transmission electron microscope (TEM) images of the Fe 3 O 4 / mesoporous SiO 2 with the size of nearly 100 nm. Plenty of pores were distributed in the entire silica layer. On the contrary, rare pores were found from Figure 2c, which was obtained by a traditional stöber method without the assistance of CTAB and TMB. The enlarged pores were beneficial to the increment of molecule-loading capacity and the penetrance of irradiation light, as well as the improvement of stability of the nanoplatform. Furthermore, the 3-mercaptopropionic acid (MPA) stabilized Mn-doped ZnS/ZnS QDs were linked onto the surface of the silica shell based on the electrostatic interaction between sulfydryl groups from the MPA and amino groups from the 3-aminopropyltrimethoxysilane (APS). Figure 2d presented the TEM image of the Fe 3 O 4 /Mn-doped ZnS/ZnS nanoplatform. The surface of the nanoplatform got much rougher than the Fe 3 O 4 /mesoporous SiO 2 , due to the modification of APS and the decoration of QDs. Due to the nontoxicity of the QDs and excellent biocompatibility of the nanoplatform, there was no need to coat another SiO 2 layer onto the surface of the nanoplatform, which was superior to the previous reports.  Figure 3a, the diffraction pattern indicated the cubic structure of Fe 3 O 4 nanoparticle, represented by the peaks (2θ) located at 30°, 35 [32,33] According to the Sherrer's formula, the average size of the Fe 3 O 4 nanoparticle was calculated at nearly 11 nm, which was consistent with their TEM images. In the X-ray diffraction pattern of Fe 3 O 4 /mesoporous SiO 2 (Figure 3b), a new distinct diffraction peak located at around 23° appeared apart from the main peak of Fe 3 O 4 nanoparticle, implying that the silica shell was efficiently coated onto the surface of the Fe 3 O 4 . As shown in Figure 3c, the X-ray diffraction pattern of the Fe 3 O 4 /Mndoped ZnS/ZnS nanoplatform was similar with these of Fe 3 O 4 / mesoporous SiO 2 , and only tiny weak diffraction peaks of Mndoped ZnS/ZnS QDs appeared due to little mass of QDs relative to that of Fe 3 O 4 /mesoporous SiO 2 .
The magnetic behaviors of the Fe 3 O 4 nanoparticle and nanoplatform were measured by vibrating sample magnetometer at room temperature (300 K) in Figure 4. It was demonstrated that all the samples exhibited excellent superparamagnetism due to the absence of remanence and coercivity. From these dates, the magnetic saturation of the Fe 3 O 4 nanoparticle was calculated to 55 emu g −1 , while that of the nanoplatform was measured to 32 emu g −1 , which was lower than the Fe 3 O 4 nanoparticle. The reason was that the mesoporous SiO 2 layer and QDs contributed major portion of the gross mass of the nanoplatform. In spite of the certain reduction of the magnetic saturation, the value of 32 emu g −1 for the nanoplatform was high enough for the application in magnetic separation. Furthermore, separation test was conducted in Figure 4 (inset), demonstrating that the nanoplatform was easily dragged onto the targeted side by an external magnet in 5 min. Figure 5 shows the fluorescence spectra of the Mn-doped ZnS/ZnS QDs and the nanoplatform samples. The concentration of the QDs in the two samples was set at a same level in order to discuss the effect from the structural change. The individual Mn-doped ZnS/ZnS QDs had an emission peak at 571 nm with a narrow FWHM (full width at half maximum). For the nanoplatform sample, a slight decrease in the fluorescence spectra was observed, which was reported in the previous literature. [34,35] There are two reasons to explain this phenomenon. One reason is that surface state of the QDs was changed from the starting thiol groups (−SH) to be chained onto the   www.advancedsciencenews.com www.particle-journal.com nanoparticle would absorb the light emitting from the QDs, named as energy transfer, leading to the reduction of the fluorescence intensity. In addition, a tiny red-shift (about 5 nm) on the emission peak of the nanoplatform sample was observed due to the increment of dielectric constant. QDs had been verified be ideal labeling probes for biomedical imaging. However, the traditional QDs decorated by organic molecule must be treated with hydrophilic group to ensure their biocompatibility, leading to the decrease of the fluorescent intensity and structural stability. In addition to be nontoxic, the surface of the designed nanoplatform was functioned with hydrophilic folic acid, effectively avoiding the problem of poor water solubility. After transfection 24 h with the nanoplatforms, Figure 6a,b shows the morphology features of the breast cancer cell (MB-MBA) with the same vision under bright-field and dark-field, respectively. Orange light emitted from the cancer cells could be easily detected from the fluorescent image (Figure 6a), demonstrating that the nanoplatform was effectively uptaken by the cancer cells. From the bright-field image (Figure 6b), the entire cellular outline was in full accord with the fluorescence image. Besides, the shape of the cancer cells still maintained fusiform-like after uptaking the nanoplatform, suggesting that the cancer cell had good cell viability and the nanoplatform had no damage to the cancer cells. Furthermore, a control experiment was conducted to ensure the fluorescent signal emitting from the QDs of the nanoplatform. Figure 6c,d exhibits the bright-field and dark-field image of the cancer cells with the treatment of Fe 3 O 4 /mesoporous SiO 2 nanoparticle. There was no fluorescent signal detecting from the dark-field image, which indicated that the labeling behavior (Figure 6b) was contributed by the QDs of the nanoplatform. These results revealed that the nanoplatform was a good alternative labeling probe in biomedical images.
It was well-known that the nanostructure with NIR (nearinfrared) absorption had gain tremendous attention, because the NIR light possessed the deep penetration into tissue and no damage to normal tissues and organs. It was necessary to evaluate the potential capability of the nanoplatform for the photothermal therapy before their application in vitro. As shown in Figure 7, displaying the curves of the temperature versus time, the laser irradiation (808 nm, 1 W cm −2 ) was employed to study the photothermal effect of the nanoplatform samples with different concentration. The real-time temperature of the sample was monitored by a thermoelectric thermometer, and every date was recorded by the average of five consecutive measurements. It was known that the cancer cells would lose activity when treated with the temperature of 42 °C for 15-60 min or over 50 °C for 4-6 min. As shown in Figure 7, the higher concentration of the nanoplatform sample, the higher changed temperature increased. For example, the raised temperature of the nanoplatform sample could reach 44 °C from 25 °C with the concentration of 2 mg mL −1 after irradiation for 15 min,   www.advancedsciencenews.com www.particle-journal.com which was high enough to destroy the targeted cancer cell. However, the temperature of the PBS solution was increased by only 3.7 °C.
To verify the feasibility of the nanoplatform in destroying cancer cells, the nanoplatform with the concentration of 2 mg mL −1 was used to treat the cancer cells under laser irradiation (808 nm, 1 W cm −2 ). After incubated with the nanoplatform for 2 h, the cancer cells were treated with the laser irradiation for 10 min. Then the cancer cells were stained, and the cell viability was evaluated. As shown in Figure 8a, over 98% of the cancer cells were destroyed by the treatment of nanoplatform and laser irradiation. In the control sample incubated with the nanoplatform but without the laser irradiation, the viability of cancer cells was close to be 100% in Figure 8b, which manifested the nanoplatform had no intrinsic toxicity to the cancer cells. These studies demonstrated that the nanoplatform had the ability to induce the targeted cancer cell apoptosis under the laser irradiation.

Conclusions
In conclusion, we successfully synthetized ferroferric oxide/ QDs nanoplatforms, possessing excellent capability for fluorescent labeling and photothermal therapy for cancer cells. The nanoplatform was prepared in terms of layer-by-layer method using Fe 3 O 4 nanoparticle as the core and mesoporous SiO 2 as the protective shell, then loaded with nontoxic Mn-doped ZnS/ZnS QDs. Notably, in order to lower mass and promote the absorption of light, template method and pore-enlarging agent were employed to generate plenty of pores on the silica shell. Due to the decoration of QDs, the nanoplatform possessed strong fluorescent behavior, thus exhibiting the potential as fluorescence labeling agents for the targeted cancer cells. Rooting in the superparamagnetism the Fe 3 O 4 nanoparticle, the nanoplatform showed excellent magnetic separation. Besides, the photothermal experiments in vitro showed that the nanoplatform possessed no toxicity and the capability to ablate the targeted cancer cells under laser irradiation. These all-in-one nanoplatforms are expected to be used in the clinical application of cancer detection and therapy.
Synthesis of Fe 3 O 4 Nanoparticle: Chemical coprecipitation method was introduced and employed to prepare Fe 3 O 4 nanoparticle, possessing the advantages of being simple, environmental friendly and high-yield. Typically, ferric chloride (3 g), iron(II) sulfide (1.7 g), and 20 mL deionized water were loaded into a 50 mL three necked round-bottom flask and degassed at room temperature for 30 min under nitrogen flow. Then the temperature was heated to 80 °C with the protection of nitrogen. Concentrated ammonia solution was added to regulate the pH to 9.8. To the mixture solution, 1 mL oleic acid was added dropwise under vigorous stirring for the decoration the nanoparticles. After completion of modification process for 2 h, the obtained Fe 3 O 4 nanoparticles were repeatedly purified with deionized water and stored in 20 mL chloroform for further characterization and use.
Preparation of Mn-Doped ZnS/ZnS Quantum Dots: The preparation of Mn-doped ZnS/ZnS QDs was performed through a simple liquid phase reaction process according to our previous report. [36] Also, the stock solutions were prepared by dissolving the precursor in a certain amount of deionized water. An amount of 5 mL zinc acetate (0.1 m), 1.  The breast cancer cells (MDA-MB-231) were maintained at 37 °C with 5% CO 2 (v/v) on glass coverslip in 12-well culture plate. Immediately after the coverage reached 60-80% on the glass coverslip, the cancer cells were washed with excess PBS solution. Then 50 µL Fe 3 O 4 /QDs nanoplatform were introduced onto the culture plate and further incubated for 2 h at 37 °C with 5% CO 2 (v/v). Finally, the glass coverslip was washed with PBS solution for three times to remove the unuptaked nanoplatforms, and then the cancer cells were prepared to detect the labeling behavior under fluorescence microscopy.
Photothermal Therapy In Vitro: On the one hand, aqueous solutions of Fe 3 O 4 /QDs nanoplatform samples were irradiated by an 808 nm laser with different concentration. The temperature of all samples was recorded, and all the dates were obtained from the average of five times measurement. On the other hand, the photothermal therapy for cancer cells was investigated. As harvested from the culture plate and coincubation with adequate nanoplatform, the cancer cells were washed with excess PBS solution. Then the cancer cells were irradiated by an 808 nm laser with the power density of 1.0 W cm −2 . After irradiated for 10 min, the cancer cells were stained to examine the live and dead cells under fluorescence microscopy.
Characterization: The crystal structure was measured by X-ray diffractometer (Rigaku-Dmax 2500) with Cu Kα (0.15 405 nm) radiation. The morphologies were inspected on TEM (JEM-1400) with acceleration voltage of 200 kV. Magnetic measurement was performed by vibrating sample magnetometer (Quantum Design) at 300K. The photoluminescence (PL) emission spectra were examined by spectrofluorimeter (Hitachi F-4500) equipped with Xenon lamp as the light source.