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Fe3O4@SiO2@Au nanoparticles for MRI-guided chemo/NIR photothermal therapy of cancer cells

Novel functionalized (biofunctionalization followed by cisplatin immobilization) Fe3O4@SiO2@Au nanoparticles (NPs) were designed. The encapsulation of Fe3O4 cores inside continuous SiO2 shells preserves their initial structure and strong magnetic properties, while the shell surface can be decorated by small Au NPs, and then cisplatin (cPt) can be successfully immobilized on their surface. The fabricated NPs exhibit very strong T2 contrasting properties for magnetic resonance imaging (MRI). The functionalized Fe3O4@SiO2@Au NPs are tested for a potential application in photothermal cancer therapy, which is simulated by irradiation of two colon cancer cell lines (SW480 and SW620) with a laser (λ = 808 nm, W = 100 mW cm−2). It is found that the functionalized NPs possess low toxicity towards cancer cells (∼10–15%), which however could be drastically increased by laser irradiation, leading to a mortality of the cells of ∼43–50%. This increase of the cytotoxic properties of the Fe3O4@SiO2@Au NPs, due to the synergic effect between the presence of cPt plus Au NPs and laser irradiation, makes these NPs perspective agents for potential (MRI)-guided stimulated chemo-photothermal treatment of cancer.


Introduction
Biocompatible Fe 3 O 4 @Au nanoparticles (NPs), where Fe 3 O 4 are superparamagnetic cores coated with Au NPs, are building agents with multiple functions for early diagnostics and for new noninvasive therapies for previously incurable diseases. This makes them promising for theranostics, where imaging and therapy modalities are integrated within a single platform. 1,2 Due to the superparamagnetic features of Fe 3 O 4 cores, the NPs can be driven in biological objects by the external magnetic eld gradient, and can thus be used for labeling of biomolecules and for drug delivery. 3 Moreover, superparamagnetic Fe 3 O 4 NPs exhibit strong magnetization in external magnetic eld, which contribute to magnetic eld perturbations. As a result the dephasing of protons is activated, leading to shortening of the T 2 time (transverse or spin-spin) relaxation of the neighboring regions. 4 Also superparamagnetic iron oxide NPs with ultrasmall size can inuence the T 1 time (longitudinal or spinlattice) relaxation. 5 This makes Fe 3 O 4 @Au NPs applicable as contrast agents in MR images. The presence of Au NPs, which have high X-ray absorption, make Fe 3 O 4 @Au NPs also promising for enhancing computed tomography imaging. 6 On the other hand, superparamagnetic Fe 3 O 4 NPs are attractive candidates for novel therapeutic approaches for cancer cell selective treatment. Thus, the superparamagnetic Fe 3 O 4 NPs can be applied in magneto-mechanical cancer treatment, where NPs actuated through the use of super low frequency AC magnetic elds and contribute to cytoskeletal disruption and subsequent cell death, can be selectively enacted upon cancerous cells, while leaving healthy cells intact. 7 Superparamagnetic Fe 3 O 4 NPs also can be applied for magnetic particle hyperthermia, where the NPs exposed to alternating magnetic elds of relatively high frequencies ($100 kHz), generate heat through Néel or Brownian relaxation, which leads to temperature increase, causing subsequent damage to the surrounding cells. 8 Au NPs demonstrate localized surface plasmon resonance, 9 based on the collective oscillation of free electrons in conduction bands. 10 These properties of gold NPs nd an application in a minimally invasive technique, which is called photothermal therapy. In this therapy photothermal agents (gold NPs) convert the laser energy to heat, which kills the cancer cells. Moreover, Au NPs are widely used as a platform for drug delivery, for example cisplatin (cPt). 11 In this case, gold NPs with immobilized cPt provide a synergetic effect in cancer treatment. 12 And this synergetic effect could also be enhanced in combination with the photothermal therapy described above. 13 Combination of magnetic iron oxide and gold NPs in the framework of one compact nanoparticle (below 40 nm), in which superparamagnetic properties of iron oxide NPs and surface plasmonic properties of gold NPs complement each other, can noticeably extend the theranostic potential of the system. However, attaching Au NPs to Fe 3 O 4 is a challenging problem. One of the effective strategy, employs an intermediate adhesion layer of amino-modied silica, which coats the Fe 3 O 4 NPs, and to which Au NPs are attached. 14,15 The latter strategy allows to use hydrophobic iron oxide NPs prepared through thermal decomposition in non-aqueous media, which are nearly monodispersive. 16,17 Silica shells also can effectively enhance biocompatibility, prevent corrosion and agglomeration of Fe 3 O 4 NPs and improve their stability in aqueous solution. The presence of silanol functional groups on the surface of the core-shell Fe 3 O 4 @SiO 2 NPs makes them dispersible in polar solvents such as water, and also provides easy further modication with other functional groups, thus enabling coupling with different drugs and biomolecules. The immobilization of cPt on the nanoparticles surface, can increase the therapeutic inuence of the drug and make its impact more controllable. However, the combination all of the three components in one system is a challenging problem.
Here we design novel, targeted cPt-based Fe 3 O 4 @SiO 2 @Au NPs ( Fig. 1), which will have an improved potential in magnetic resonance imaging (MRI)-guided chemo-photothermal stimulated treatment of cancer. For this purpose, our magnetic NPs were functionalized by cPt, a metallic platinum coordination compound with a square planar geometry, which shows high anticancer activity in a variety of tumors, and which is widely used in chemotherapy medication to treat a number of cancers. 18 For the theranostic application, we tested the effectiveness of the NPs as contrast agents for MRI and investigated in vitro the chemo-phototherapy potential of the Fe 3 O 4 @-SiO 2 @Au nanosystem on two colon cell lines: SW480 and SW620 in terms of cell viability (MTT assay) and morphological changes induced in tumor cells (optical microscopy). Because near-infrared (NIR) radiation can penetrate into biological tissues several centimeters deep, we chose a laser with an 808 nm wavelength, which is employed in photothermal therapy in the majority of cases. 19 The morphology, structure and magnetic properties of the Fe 3 O 4 @SiO 2 and Fe 3 O 4 @-SiO 2 @Au nanosystems have been investigated using transmission electron microscopy (TEM), X-ray diffraction (XRD), Mössbauer spectroscopy and SQUID-magnetometry. The stability of the functionalized Fe 3 O 4 @SiO 2 @Au NPs and the verication of the effective process of biofunctionalization and cPt immobilization have been carried out using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and Raman spectroscopy.

Materials
Tetraethyl ortho-silicate (TEOS, 99.0%), (3-aminopropyl)triethoxysilane (APTES, 99%), polyoxyethylene (5) 16,17 Shortly, in rst step an ironoleate complex was prepared. For this purpose, a solution of iron chloride (FeCl 3 $6H 2 O, 3.6 g) and sodium oleate (12.2 g) dissolved in a mixture of ethanol (27 mL), hexane (47 mL) and distilled water (20 mL) in a 250 mL necked round bottom ask was prepared. The above-mentioned solution was heated to 70 C with a constant heating rate of 3.3 C min À1 under Ar ow and was kept at that temperature for 4 hours. Aer the reaction was complete the upper red brown layer of iron oleate was washed three times with distilled water in a separatory funnel and subsequently dried in air. In the second step, the synthesized iron-oleate complex (9 g) with oleic acid (1.43 g) were dissolved in 1-octadecene (63 mL) at room temperature. The solution placed in a 250 mL necked round bottom ask was heated with a constant heating rate of 3.3 C min À1 to reux (318 C) under argon for 30 min. At the end of the reaction the brownish-black solution was cooled to room temperature and the obtained NPs were precipitated in ethanol (200 mL) and centrifuged for 5 minutes in 50 mL acons at 4000 rpm. Then the NPs were re-dispersed in chloroform and isolated by adding acetone and centrifuging the obtained solution. This washing step was repeated two times and the third time the NPs were le in a chloroform/acetone mixture for 24 hours. The NPs were subsequently collected by centrifugation, dried, re-dispersed and stored in cyclohexane, where the concentration of NPs was set to 2.5 g L À1 .
(b) Fe 3 O 4 @SiO 2 NPs. The Fe 3 O 4 @SiO 2 complexes were fabricated by reverse microemulsion method. 20 Briey, Igepal CO-520 (1.0 g) was dispersed in cyclohexane (22 mL) and sonicated for 10 min. Then, 25%-ammonium hydroxide (0.4 mL) was added to 1 mL of the as-prepared Fe 3 O 4 NPs cyclohexane solution and stirred for 30 min. Subsequently, TEOS (0.07 mL) was added to the above mixture and the nal solution was le under continuous stirring for 12 hours. Aer this, APTES (0.05 mL) was dropped into the mixture, which was le under continuous stirring for another 12 hours. The obtained Fe 3 O 4 @SiO 2 core-shell NPs were collected by centrifugation and washed with ethanol three times and re-dispersed in 1 mL of ethanol.
(c) Fe 3 O 4 @SiO 2 @Au NPs. The procedure of Fe 3 O 4 @SiO 2 NPs decoration by Au NPs can be divided into two steps. The rst step is the synthesis of Au NPs and the second step is attaching the Au NPs on the surface of Fe 3 O 4 @SiO 2 , which is functionalized with APTES. Au NPs with diameters from 2 nm to 6 nm were synthesized by direct reduction of HAuCl 4 $3H 2 O with THPC previously reported in the literature. 21 Briey, portions of sodium hydroxide (1 mol, 0.5 mL), the reducing agent THPC (1% aqueous solution, 1 mL), and the metal salt HAuCl 4 (1% aqueous solution, 2 mL) were added successively to 38 mL of water while stirring and the resulting solution was aged for a few days at 4 C, till orange-brown hydrosols of gold were formed. Then the solution of Fe 3 O 4 @SiO 2 NPs (0.5 mL) was mixed with the 5 mL of gold seed solution. The obtained Fe 3 -O 4 @SiO 2 @Au NPs were collected by magnet and washed with water and ethanol for two times.

Biofunctionalization of the Fe 3 O 4 @SiO 2 @Au NPs and immobilization of cPt on their surface
In order to attach the drugs to the stabilized Fe 3 O 4 @SiO 2 @Au NPs, 16-MHDA was used as a linker. This chemical compound has 16 carbon atoms, a thiol group (-SH) on one side and a carboxyl group on the other side. Thanks to the thiol group, the 16-MHDA is linked with the NPs surface and the COOH group is linked with cPt. Moreover, using this linker, the active surface of Au NPs was not coated, as it is in the case when e.g. polyethylene glycol (PEG) shell was used. First, the NPs were incubated with MHDA for overnight at 4 C. Aer rinsing with DMF, the MHDA-covered NPs-structures were incubated in DMF solution of PFP, DIPEA and CMC, during 30 min at 25 C. Aer repeated rinsing with DMF and centrifugation, a cPt solution was added and incubated for 30 min at 25 C. The scheme of functionalization (biofunctionalization and immobilization of cPt on their surface) of Fe 3 O 4 @SiO 2 @Au NPs is shown in Fig. 1.

TEM characterization
The morphology of the fabricated NPs was investigated by scanning transmission electron microscopy (STEM) using the high-angle annular dark-eld detector (HAADF). Selected area electron diffraction (SAED) patterns were taken in the TEM mode. Energy dispersive X-ray spectroscopy (EDS) was used to analyze the chemical composition of the synthesized NPs. All these measurements were performed on an aberrationcorrected FEI Titan electron microscope operating at 300 kV equipped with a FEG cathode. The particle size distribution was evaluated based on the HRSTEM images taken from different areas of the TEM grids.

X-ray diffraction
The crystal microstructure of the fabricated NPs was analyzed using a two-circle laboratory diffractometer Panalytical X'Pert Pro. The measurements were performed in the standard q-2q geometry using Cu K a radiation (l ¼ 0.15406 nm). The dispersion of the NPs was dried on a zero-background holder and was placed on a sample spinner with rotation time adjusted to 16 seconds. The data were collected in the range between 25-85 (2q) with a step size of 0.08 .

Mössbauer spectroscopy
Mössbauer spectra were recorded in transmission geometry at room temperature and at 16 K using the spectrometer MS4 (SeeCo, USA) with a 57 Co/Rh source (12 mCi). Low temperature measurements were performed using a closed cycle refrigerator system CCS-850 (Janis Research Company, USA). The temperature was controlled using a Lakeshore temperature controller LS335 (Lake Shore Cryotronics, Inc., USA) with dual calibrated DT-670 sensors with an accuracy of AE0.005 K. The spectra were tted using the MOSMOD soware, assuming a Gaussian distribution of the hyperne magnetic eld (H hf ) and quadrupole splitting within the iron nuclei. 22 Lorentzian line shape of source natural line width was determined from the Mössbauer spectrum of a pure 28 mm thick a-Fe foil. The spectrometer was calibrated to pure a-Fe by collecting the spectra of a standard a-Fe foil at room temperature and 16 K. All isomer shis (IS) are presented in respect to an a-Fe standard.

Magnetometry
A SQUID-magnetometer (MPMS XL, Quantum Design) was used to investigate the magnetic properties of iron-oxide NPs. The zero-eld cooled (ZFC) temperature dependences of the magnetization were measured by cooling the sample from 300 K to 5 K and then applying a eld of 100 Oe to record the magnetization, while the sample was heated from 5 K to 300 K. The eld cooled (FC) measurements were made with a eld of 100 Oe applied during the cooling process. The hysteresis curves were measured at 5 K and 300 K, respectively.

UV-Vis measurements
The UV-Vis measurements were performed with a Lambda Bio20 instrument from PerkinElmer. The resolution was chosen to be 1 nm and the scan speed was 240 nm min À1 . The spectral range was from 200 nm to 800 nm.

MRI
To investigate the effectiveness of the NPs as contrast agents, MR imaging as well as relaxometry were performed using a 9.4T Bruker Biospec 94/20 MRI scanner. For the acquisition of the series of axial images of each sample, the RARE with variable repetition time T R (RAREVTR) and the MSME imaging sequences were used. Application of RAREVTR sequence allows to obtain simultaneously T 1 and T 2 relaxation times maps of samples with different concentrations of the NPs. MSME sequence was employed in order to increase the number of echoes and therefore to obtain better quality of T 2 measured and T 2 -weighted images. For acquired T 1 and T 2 values, linear regression of relaxation rates: vs. NPs concentration in samples was performed: where: Cthe concentration of NPs, R 1,2 the relaxation rate of a whole sample, R 1S,2Sthe relaxation rate of a solvent without contrasting agent, r 1,2the relaxivity of Fe 3 O 4 NPs.

FTIR measurements
The Fourier transform infrared absorption (FT-IR) spectra in the wavelength range between 400-4000 cm À1 were acquired using an EXCALIBUR FTS-3000 spectrometer at room temperature and measured for the respective samples mixed with KBr. The sample was dried and sandwiched between two KRS-5 window disks. The 64 scans were averaged at a resolution of 4 cm À1 .
During the experiments, the spectrometer was purged with dry nitrogen. Baseline correction and normalization of FTIR spectra were applied.

Calorimetric and TGA measurements
Calorimetric measurements were carried out under dry 5.0 pure nitrogen purge (25 mL min À1 ) on a TA Instruments' DSC 2500 differential scanning calorimeter equipped with a liquid nitrogen LN2P cooling pump. Thermal behaviour of samples placed in aluminium pans and crimped with hermetic lids, was studied in a temperature range from 5 C to 250 C with a maximum heating rate of 5 C min À1 . Calibrations of the temperature and enthalpy were performed using an indium standard. Additionally, modulated differential scanning calorimetry was employed to gain more thorough insight into thermal properties of the specimens. TGA experiments were carried out under owing synthetic air (N 5.0 ) on a TA Instruments' TGA 5500 high-resolution thermogravimetric analyser using platinum 100 mL pans with a maximum heating rate of 5 C min À1 . The specimens were either directly placed in open 100 mL platinum pans or previously enclosed inside aluminium pans to limit the evaporation rate and measured in a temperature range from the ambient temperature to 500 C. The temperature calibration was carried out using nickel and alumel standards. Additionally, high resolution (HiRes TGA) mode was employed to gain more precise values of the experimental points.

FT-Raman spectroscopy
FT-Raman spectra were recorded using a Nicolet NXR 9650 FT-Raman Spectrometer equipped with an Nd:YAG laser (1064 nm) and a germanium detector. The measurements were performed in the range from 150 cm À1 to 3.700 cm À1 with a laser power of 1 W. The unfocused laser beam was used with a diameter of approximately 100 mm and a spectral resolution of 8 cm À1 . Raman spectra were processed by the Omnic/Thermo Scientic soware based on 128 scans.

Cell lines
As the in vitro model, two human colon cancer cell lines (SW480 and SW620) were used, which were obtained due to the courtesy of Prof. Caroline Dive, Paterson Institute for Cancer Research, University of Manchester. These cell lines were cultured in DMEM with high glucose (Corning, NY, USA) in a 37 C humidied atmosphere with 5% CO 2 . All media were supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaille, France) and gentamicin (50 mg mL À1 ), (PAN-Biotech, Aidenbach, Germany). The cells were cultured by bi-weekly passages and were regularly tested for Mycoplasma sp. contamination by PCR-ELISA kit (Roche, Mannheim, Germany) according to the manufacturers' instruction.

Light microscopy images of cells
The images of cells at 100Â magnication were taken using Microscope Olympus IX70 (Olympus Corporation, Tokyo, Japan).

Light source and cells irradiation protocols
The irradiation of the cells was conducted by a low-intensity laser operating at 808 nm wavelength. An adjustable power supply was connected to the setup to enable control of the power output of the laser with intensity of 100 mW cm À2 and irradiation time of 5 minutes. The description of the all samples, which were studied in this experiment, is presented in Table 1.

Analysis of cell viability data
The obtained MTS assay results are represented as the means AE SEM (the standard error of the mean). The quantitative results were nally compared with the T test. P value < 0.05 was considered to be statistically signicant. Moreover, in the MTS test, data for each treatment point shows an average from three parallel wells. The data were analyzed and presented graphically using the Origin Lab2019b soware.

Results and discussion
Characterization of nanoparticles    Fig. 2c and f, where a well-dened core-shell structure with one iron-oxide core surrounded by a homogeneous SiO 2 shell is observed. The average thickness of the shell is around 10 nm (Fig. 2i). In the third stage the Fe 3 O 4 @SiO 2 core-shell NPs were decorated with Au NPs with an average size of 4 nm, Fig. 2d, g and j. From Fig. 2d and g, it can be seen that every Fe 3 O 4 @SiO 2 core-shell nanoparticle has a few Au NPs on its surface. The UV-Vis absorption spectra of different Fe 3 O 4 NPs with and without a SiO 2 shell as well as Fe 3 O 4 @SiO 2 decorated by Au NPs are presented in Fig. 3. In the UV-Vis spectrum of pure Fe 3 O 4 NPs, no obvious peaks were observed. However, encapsulation of the magnetic NPs in a 10 nm SiO 2 shell, caused the appearance of a peak at 250 nm in the spectra of the Fe 3 O 4 @-SiO 2 NPs. This peak corresponds to the created core-shell structure of the NPs, and may originate from the changes of the band gap caused by the quantum size effect and surface effect of nanostructures, 24 as well as from the Fe-O-Si bonds of the coreshell NPs. 25 Moreover, decorated Fe 3 O 4 @SiO 2 NPs with 4 nm Au NPs caused the appearance of a broad peak around $540 nm, which originates from the gold NPs. 26 Interestingly, the peak at 250 nm, which was visible in the UV-Vis spectrum of Fe 3 O 4 @-SiO 2 , was shied towards lower wavelengths. This can suggest, that the synthesis of Au NPs on the Fe 3 O 4 @SiO 2 NPs surface caused changes in the Fe-O-Si bonds, which are, as most number of chemical bonds, sensitive for the local environmental changes. 25 In the FTIR spectra of pure Fe 3 O 4 NPs, Fe 3 O 4 @SiO 2 NPs and Fe 3 O 4 @SiO 2 @Au (Fig. 4) a peak at 590 cm À1 corresponding to Fe 3 O 4 is observed. Moreover, peaks at 1110 cm À1 , 920 cm À1 , 790 cm À1 , 670 cm À1 originating from FeOH are noticed. Furthermore, peaks characteristic for g-FeO(OH) and a-FeO(OH) are visible in FTIR spectra of Fe 3 O 4 NPs at 1026 cm À1 , 1161 cm À1 , 753 cm À1 and 890 cm À1 , 797 cm À1 wavenumbers, respectively. 27 In the FTIR spectra of Fe 3 O 4 @SiO 2 NPs and Fe 3 -O 4 @SiO 2 @Au a peak at 1560 cm À1 corresponding to C-N vibration is visible. The presence of these peaks indicates that the Fe 3 O 4 NPs were successfully modied by the SiO 2 groups, thus forming Fe 3 O 4 @SiO 2 nanoparticles. 28 The crystalline structure of the NPs fabricated at every stage of the synthesis was investigated using XRD (Fig. 5). The XRD pattern of Fe 3 O 4 was rened with the Rietveld method using the Fullprof soware, 29 where for peaks tting a Voigt function in the Thompson-Cox-Hastings approximation was applied. 30 The prominent peaks at 30.17 (220), 35    in the Fullprof program was a ¼ 8.361(1)Å. The obtained value is the average value of the lattice constant for bulk magnetite (a ¼ 8.39Å) 31 and for bulk maghemite (a ¼ 8.33Å), 32 what is typical for Fe 3 O 4 NPs. 33 The XRD pattern of the Fe 3 O 4 @SiO 2 core-shell NPs looks almost the same as the pattern for pure Fe 3 O 4 NPs, except the broad peak, which originates from the amorphous SiO 2 . The XRD pattern of the Fe 3 O 4 @SiO 2 @Au NPs contains extra wide peaks, which can be assigned to the (111), (200), (220), (311) and (222) diffraction peaks of small Au NPs with fcc structure. 34 This allows to suggest that the encapsulation process of Fe 3 O 4 in the SiO 2 shell has no inuence on the initial structure of the Fe 3 O 4 NPs, which does also not change during the Au NPs deposition, due to good protection function of the SiO 2 shell. 35 In order to ensure that the encapsulation into the SiO 2 shell has no signicant inuence on the initial magnetic properties of the Fe 3 O 4 cores, and to evaluate the application potential of the investigated nanosystems for magnetic resonance imaging, we made magnetometry of the pure Fe 3 O 4 NPs and the Fe 3 O 4 @SiO 2 core-shell NPs at room temperature (300 K) and at 5 K ( Fig. 6a and b). As seen from the TEM images (Fig. 2), the SiO 2 shell entirely covers the nanoparticle and protects the iron-oxide core from the environment, preventing its further oxidation. This means that possible changes of magnetic properties of the NPs may occur only at the stage of Fe 3 O 4 NPs encapsulation into SiO 2 shell, when the iron-oxide core can be oxidized. Also, as can be seen from Fig. 2b and c, the SiO 2 shell is the main reason for the relative decrease of iron oxide content in the particles, which will inuence the total value of saturation magnetization of the NPs. It was found that at 300 K, pure and core-shell NPs exhibited superparamagnetic behavior with no evident coercivity or remanence. 38 For Fe 3 O 4 @SiO 2 NPs saturation magnetization is lower (M S (300 K) ¼ 15 emu g À1 ) compared to pure NPs (M S (300 K) ¼ 46 emu g À1 ). The reduced saturation magnetization of the Fe 3 O 4 @SiO 2 NPs, resulting in decreased magnetic anisotropy and attributed to the relative decrease of iron oxide content in the particles, is mainly explained by the presence of the silica shell coating. 39 It also worth mentioning that in the case of pure Fe 3 O 4 NPs, the measured values of M S can be reduced by the presence of the protective shell of oleic acid, which prevents the interaction of iron-based NPs with the atmosphere. 33,40 The temperature dependence of the magnetic moment M(T) of the Fe 3 O 4 and Fe 3 O 4 @SiO 2 NPs under an applied magnetic eld (H ext ¼ 100 Oe) aer zero-eld cooling (ZFC) and aer eld cooling (FC) was measured from 300 K to 5 K (Fig. 6c). This dependence allows to determine the blocking temperature (T B ) of the NPs, being the temperature above which the majority of the particles becomes superparamagnetic, and above which the FC and ZFC magnetization curves coincide. For   decomposition method, the exchange bias elds originate from the presence of trace amounts of wustite (FeO) and thus this higher content of FeO is responsible for the larger exchange bias elds. 42 The matching of the values H EB and H C allows to conclude that encapsulation of Fe 3 O 4 cores by the SiO 2 shells does not inuence the structure of the Fe 3 O 4 cores. As it can be expected, the attachment of small Au gold NPs does not inuence the magnetic properties of the Fe 3 O 4 NPs and Fe 3 O 4 @SiO 2 @Au NPs can be easily attracted by a magnet (Fig. 6d).
For local structure characterization of the as fabricated Fe 3 O 4 cores and aer their encapsulation in the SiO 2 shell, Mössbauer spectroscopy measurements were performed. Fitted 57 Fe Mössbauer spectra are shown in Fig. 7, while hyperne parameters of sub-spectra and their relative contribution (area ratio) are summarized in Table 2. It is established by Chlan et al. 43 and Senn et al., 44 that above the Verwey transition temperature (125 K), stoichiometric magnetite has a cubic Fd 3m symmetry with inverse spinel cubic structure. The cell unit (Fe 3+ ) A (Fe 3+ Fe 2+ ) B consists of Fe 3+ ions at the Asite (tetrahedral) and Fe 2+ ions at the B-site (octahedral). At tetrahedral sites, Fe atoms are in a +3 state, while in B positions the mean valence is +2.5. Mössbauer spectra of the as fabricated Fe 3 O 4 core samples and aer their encapsulation in the SiO 2 shell at room temperature ( Fig. 7a and c), are characterized with singlet IS ¼ 0.32 mm s À1 , without resolved quadrupole splitting corresponding to Fe 3+ ions. The observed broadened baseline in spectrum of NPs, is most probably caused by the presence of an unresolved magnetic sextet with weighted average isomer shi $0.6 mm s À1 corresponding to Fe 2.5+ ions, which indicates a superparamagnetic state. At low temperatures, the structure of magnetite is monoclinic with a Cc space group symmetry. 44 Aer Verwey transition, the unit cell becomes four times bigger than cubic, doubled in the c direction, and with Fe ions residing in 16 B inequivalent positions (8 Fe 3+ and 8 Fe 2+ ) and 8 A inequivalent positions. 43 The spectrum of Fe 3 O 4 NPs (Fig. 7b)  ions at A site to the contribution of sextets of Fe ions at B site is 0.56, which is very close to the reference value of stoichiometric magnetite. 45,46 At the same time the relative contribution of subspectrum attributed to Fe 2+ ions at B site is less than expected (only 17% against $30% expected). The lower contribution of Fe 2+ ions in Mössbauer spectrum could be due to the partial oxidation of Fe 3 O 4 NPs leading to the formation of some amount of maghemite or wustite at the surface of NPs. The Mössbauer spectra of the Fe 3 O 4 cores aer their encapsulation in the SiO 2 shell (Fig. 7d) are very similar to those of the Fe 3 O 4 NPs, but with slightly smaller values of IS and H hf . As it was mentioned by Lyubutin et al., 47 the shell material can induce pressure on the NPs and this effect can change the Mössbauer parameters. From the other side, the shi of IS and H hf values may be attributed to the formation of interfacial structure. 48 The above-mentioned results conrm that the asfabricated Fe 3 O 4 NPs and those encapsulated by the SiO 2 shell possess a very similar crystal structure.

MR imaging of Fe 3 O 4 @SiO 2
In order to evaluate the theranostic potential of the studied nanoparticles, we investigated the MRI enhancing function of the active element of the nanoparticle (i.e. Fe 3 O 4 @SiO 2 ), which can inuence the MRI contrast. In this experiment we investigate only the active element of the nanoparticle because it was the only component exhibiting strong magnetic properties, causing local perturbations of the magnetic eld, which resulted in signal loss due to signicant shortening of the transversal relaxation time T 2 . In addition, neither Au nanoparticles (see Fig. 2g) nor MHDA linker (see Fig. 1) form a continuous shell around the Fe 3 O 4 @SiO 2 NPs. As a result, the Au NPs and attached drugs did not interfere to prevent water molecules from possible direct contact with the surface of the active element. Alternative behavior was observed for the case of continuous shell observed for NPs studied, for example, by Hou et al. 14 where Fe 3 O 4 @SiO 2 NPs were covered by 15 nm gold continuous shell. In our case the water molecules were very close to the surface of Fe 3 O 4 @SiO 2 NPs and the dephasing of protons was activated by magnetic eld perturbations in the direct vicinity of the Fe 3 O 4 @SiO 2 NPs. Thus, for MRI contrasting properties evaluation, we calculated the values of the relaxation rates: R 1 and R 2 from the measured relaxation times for Fe 3 O 4 @SiO 2 active element of the NPs. The dependences of the relaxation rates R 1 and R 2 on the concentration of Fe 3 -O 4 @SiO 2 nanoparticles are presented in Fig. 8a and b respectively. The effect of increasing NPs concentration on the relaxation rate is signicantly higher for R 2 , especially in low concentration range, most interesting for potential clinical application of the theranostic agents. In a concentration range up to 0.1828 mmol L À1 , there was a very good linear dependence of nanoparticles concentration on the R 2 relaxation rate (R 2 above 0.99). For higher concentrations, the effect of T 2 shortening was less pronounced, which can be attributed to clustering of NPs, as their concentration in the solution increases. 49 This effect of NPs clustering was observed visually in samples with the highest concentration (Fe 3 O 4 concentrations: 0.7311 and 0.3655 mmol L À1 ), therefore the specic relaxivity r 2 was determined only for unclustered NPs. Linear regression results for R 2 relaxation rates were: R 2S ¼ 1.894 AE 0.033 s À1 , r 2 ¼ 579.5 AE 4.3 s À1 mmol À1 L. Conversely, the effect of T 1 shortening was practically not observed for low NPs concentrations up to 0.0457 mmol L À1 , with T 1 values characteristic for distilled water, and in higher concentration range, it was much weaker than observed for T 2 . This effect was conrmed by other papers 5,14 and can be attributed to limited access of water molecules to superparamagnetic center (caused by SiO 2 coating), which is crucial for r 1 enhancement. Only signicantly higher concentrations of Fe 3 O 4 @SiO 2 NPs, provide enough superparamagnetic centers to effectively change the T 1 relaxation time of the whole sample. 50 Linear regression results for R 1 relaxation rate were: R 1S ¼ 0.3113 AE 0.0020 s À1 , r 1 ¼ 0.5665 AE 0.0070 s À1 mmol À1 L, illustrating three orders of magnitude difference between r 2 and r 1 relaxivities.
The obtained results showed that the examined nanocapsules exhibit a strong effect of R 2 relaxation rates. In lower concentration range, which is more interesting in view of possible clinical applications, the effect of T 1 relaxation time is very small, and will have negligible effect on the MR image contrast change. Therefore, the investigated Fe 3 O 4 @SiO 2 nanoparticles, exhibit very strong T 2 contrasting properties ("negative" contrast), which is greatly visible in T 2 -weighted images (see Fig. 8c and d). Here, the MR images for high and low NPs concentration ranges are compared separately, to illustrate the possibility of effective contrasting in both cases. For high concentrations however, much shorter echo time (T E ) in MRI pulse sequences have to be applied to get reasonable images.

Stability of the biofunctionalized Fe 3 O 4 @SiO 2 @Au NPs with immobilized cPt on their surface
In order to evaluate the stability of the functionalized Fe 3 -O 4 @SiO 2 @Au NPs and to verify the effectiveness of the biofunctionalization and immobilization process, Raman spectroscopy, as well as TGA and DSC analysis, have been performed.
In this study, we show a successful biofunctionalization of Fe 3 O 4 @SiO 2 @Au, which is very important for an effective delivery of the drug to cancer cells. For this purpose, Raman spectra of four samples i.e. cPt (Fig. 9, black spectrum), pure MHDA (Fig. 9, red spectrum), the MHDA capped Fe 3 O 4 @-SiO 2 @Au NPs (Fig. 9, blue spectrum) and Fe 3 O 4 @SiO 2 @Au NPS + MHDA + cPt (Fig. 9, green spectrum) were acquired. In the Raman spectrum of the MHDA sample a peak of the thiol group (-SH) at 2900 cm À1 is visible. 51 The disappearance of this peak in the biofunctionalized and cPt containing samples conrms a successful attachment of the MHDA to the surface of gold NPs. Moreover, in the Raman spectrum of Fe 3 O 4 @SiO 2 @Au + MHDA + cPt (Fig. 9, green spectrum), a peak corresponding to the C]O vibrations (1680 cm À1 ) is observed. 52 This group is responsible for linking cPt with biofunctional surfactants on the Fe 3 O 4 @-SiO 2 @Au surface. 51 Both observations provide an evidence on  The results of TGA experiments presented in Fig. 10 a, were measured for liquid functionalized Fe 3 O 4 @SiO 2 @Au NPs and were acquired either directly in an open pan or using a hermetic aluminium container with a hermetic lid, automatically punctured right before loading the sample into the TGA furnace. While the curve registered in the conventional TGA mode shows only strong evaporation of the solvent, the data recorded during the HiRes mode experiment (solid line), indicate two major weight loss regions. First, a small, but still pronounced decrease of mass starts slightly above 93 C (7.14% weight loss), and the second, much larger one, follows from ca. 103 C (92.8% weight loss). Most probably both observed effects are due to the conversion of the solvent itself, rather than of the active pharmaceutical ingredient.
The DSC thermogram registered upon heating is dominated by a broad peak starting around 147 C, originating from an endothermic process reecting a decomposition of the sample. Nevertheless, a group of small thermal anomalies is also visible between 95 C and 122 C (Fig. 10b). A closer inspection of the heat ow curve measured during the MTDSC experiment (inset in Fig. 10b), revealed one exothermic thermal effect followed by two endothermic ones. Further analysis of the heat ow signal allowed to conclude that these anomalies are strictly kinetic, since they are contributing to the non-reversing (kinetic) component of the measured heat ow.
The results of both TGA and DSC measurements, indicate that the specimen is thermally stable at least up to 90 C. However, further study of the anomalies revealed during DSC and TGA experiments above 90 C are necessary and might shed new light on decomposition kinetics of the functionalized Fe 3 -O 4 @SiO 2 @Au NPs.
Simulation of combined chemo-phototherapy of colon cancer cells Morphology of colon cancer cells. The morphological analysis of the two lines of colon cancer cells (Fig. 11), provides information about the inuence of functionalized Fe 3 O 4 @-SiO 2 @Au NPs and laser irradiation effects on the cells. Detailed information about the morphology changes in cells cultured with functionalized and non-functionalized NPs, with cPt, with and without irradiation are presented in the ESI in Fig. S1. † The changes of the morphology of cells were compared with the control SW480 and SW620 cell lines presented in Fig. 11a1 and b1, respectively. Light microscopy images of SW480 and SW620 cells cultured with Fe 3 O 4 @SiO 2 @Au + cPt and irradiated with an 808 nm laser, showed the largest changes in the morphology and the smallest number of living cells (Fig. 11a2 and b2). In comparison with the control, only small differences in the morphology of cells from both cell lines were visible in the microscopy images of cells cultured with non-functionalized nanoparticles ( Fig. S1a1 and b1 †). These cells had a spherical shape and evidently adhered to the substrate. Strikingly, the addition of functionalized nanoparticles to the cells and their subsequent irradiation, showed a strong effect on both: cell morphology and cell death. It was related to the immobilization of cPt on the surface of NPs and the presence of the small Au NPs at the surface of the Fe 3 O 4 @SiO 2 NPs. Cisplatin involves creating a cross-linking between adjacent DNA strands and  This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 26508-26520 | 26517 within the same strand. The formation of these crosslinks prevents DNA replication and cell division. 53,54 Moreover, the presence of Pt in the structure of cPt causes, that this drug has catalytic properties, which are better at high temperatures. 55 The high temperature is induced by the laser irradiation of the 4 nm Au NPs, which absorb light and subsequently they convert the light energy into heat energy. This conversion is caused by several photo-physical processes, which take place one aer the other. 56 Firstly, light absorbed by Au NPs is quickly converted to heat and forms a hot metallic lattice by two processes: electronelectron relaxation, which is observed in femtoseconds and electron-phonon relaxationpicoseconds. During these processes, the temperature in the Au NPs could increase to about 1000 K. When the light beam is turned off, the phononphonon relaxation is stopped and the heat energy goes to the cells. 57 The cancer cells are sensitive to high temperatures, therefore, the laser irradiation combined with Au NPs causes death of cells, which was observed in the Fig. 11a2 and b2 as cells were not sticking to the ground. Moreover, the small size of Au NPs caused, that 100% of the light energy was converted into heat energy. 58 Viability. The viability test showed, that the smallest cytotoxic effect for both SW480 and SW620 cell lines was observed for the C@808, $2% and C@cPt, 4% and 2% samples. Moreover, the MTS assay showed a relatively small decrease of viability of cancer cells cultured with the Fe 3 O 4 @SiO 2 @Au NPs. The small cytotoxicity of Fe 3 O 4 @SiO 2 @Au NPs can be caused by the medium, in which the cells were cultured, because this medium reduces the repulsive forces among the NPs, therefore aggregation of Fe 3 O 4 @SiO 2 @Au NPs, was possible. 59 However, a certain toxicity effect was observed in the MTS results, Fig. 12.
It can be caused by the interaction between the nanoparticles and the proteins, which are in the cell membranes. 60 Furthermore, the accumulation of nanoparticles, as well as their cytotoxicity effect is dependent not only from their size or shape, but also from cells line, which was cultured with the nanoparticles. 61 Therefore, in our study we observed differences in the viability between the cells from different lines. Moreover, the observed toxicity differences between the cell lines, could be caused by the fact that the SW480 cell line originates from the primary tumor, whereas the SW620 line is from a metastatic lesion to the lymph node, although both cell lines originate from the same patient. The metastatic lesion cells are very oen more aggressive than cells from the primary tumor. 62 Laser irradiation of the cells cultured with cPt caused 12% and 8% mortality of the SW480 and SW620 cell lines, respectively, which is around three and four times higher, than the mortality effect of cells cultured with cPt, but not subjected to irradiation.
Cells cultured with non-functionalized Fe 3 O 4 @SiO 2 @Au NPs and irradiated by laser, exhibited a very high mortality in comparison with other samples. This effect was caused by the Au NPs decorating the Fe 3 O 4 @SiO 2 NPs. Gold NPs show photothermal effects of plasmon-resonance, which depend, among others, on the NPs size. Therefore, it can be assumed, that Au NPs were used as photosensitizers in this study. The temperature effects, correlated with the nanoparticle sizes were theoretically explained by Jain et al. 63 They showed the relationship existing between the size of the Au NPs diameter and the optical absorption or scattering properties of these nanoparticles using the Mie theory. Indeed, for the gold nanoparticles with $20 nm diameter, the main contribution to the total extinction of light was absorption. Interestingly, with the increase of the nanoparticle size up to $40 nm, the values of light extinction were dependent on the scattering effects. When the particle size was $80 nm, the extinction of gold nanoparticles was caused by both, absorption and scattering. 64 Moreover, Jiang et al. calculated, using experimental data as well as the Mie theory, the dependence between the particle size and photothermal conversion efficiency of spherical gold nanoparticles and they showed, that smaller nanoparticles can more efficiently transduce the energy from light to heat than larger Au NPs. 65 The highest, $50% and $43% mortality of the SW480 and SW620 cells were observed in the case of C@808cPtNPs samples. This could be caused by synergistic effect of both components cPt and Au NPs, as well as the enhancement by laser irradiation. 66

Conclusions
In conclusion, a new type of compact Fe 3 O 4 @SiO 2 @Au nanoparticles (below 40 nm in size) for theranostic applications was designed, synthesized and investigated. The fabricated nanoparticles were uniform, well dispersed in water and had low distribution in sizes of the spherical Fe 3 O 4 core (z11 nm), the thickness of SiO 2 shell (z10 nm), and Au nanoparticles (z4 nm) deposited on SiO 2 shell. The investigated nanoparticles showed pronounced superparamagnetic properties at room temperature with high saturation magnetization, which is Fig. 12 Viability of colon cancer cells: SW480 (a); SW620 (b) after addition of cPt, Fe 3 O 4 @SiO 2 @Au, functionalized Fe 3 O 4 @SiO 2 @Au and laser irradiation in the presence of these three systems. Data was considered as significant when *p < 0.05 vs. control. caused by Fe 3 O 4 core. The superparamagnetic state was conrmed by Mössbauer spectroscopy, which showed a short relaxation time of the NPs magnetic cores. It was found that the structure and magnetic properties of the Fe 3 O 4 core were stable and were very similar before and aer their encapsulation in the SiO 2 shell, which stabilized them and made dispersible in water. The studied nanoparticles exhibited very strong T 2 contrasting properties, which is nicely visible in T 2 -weighted images, and which made the nanoparticles applicable in diagnostics, as contrast agents in MRI. The investigated nanoparticles also demonstrate low toxicity, because of the biocompatible SiO 2 continuous shell, which reliably prevents the contact of the Fe 3 O 4 magnetic core with the environment. Moreover, functionalized nanoparticles showed a synergetic effect of investigated nanoparticles and anticancer drugs in chemo-photothermal stimulated treatment of cancer. All of the as mentioned properties of the nanoparticles showed their potential in magnetic resonance imaging (MRI)-guided chemophotothermal stimulated treatment of cancer.

Conflicts of interest
There are no conicts to declare.