Novel Carbon-Based Magnetic Luminescent Nanocomposites for Multimodal Imaging

Multifunctional nanocomposites can combine multiple functions into a single nanosystem and thus have attracted extensive interest in various fields. The combination of magnetic and upconversion luminescent nanoparticles into one single nanoplatform, which have a good application in biomedical fields such as bio-magnetic separation, magnetic resonance imaging (MRI), and optical imaging, is highly desirable. Here we reported multifunctional nanocomposites by using hollow carbon sphere to integrate magnetic Fe₃O₄ and upconversion nanoparticles (UCNPs) into one nanosystem. The as-prepared UCNPs/Fe₃O₄@h-C have near-infrared (NIR) luminescence under 980 nm excitation and superparamagnetism. In addition, since the carbon layer can absorb NIR light and transfer it into heat with high efficiency, the nanocomposites can realize photo thermal (PT), upconversion luminescence (UCL) and MRI tri-mode imaging. The UCNPs/Fe₃O₄@h-C might be further utilized as a potential theranostic agent, including its in-depth monitoring through luminescent imaging and MRI diagnosis, as well as its direct use in tumors as a photothermal therapy (PTT) agent.

Introduction

In recent years, multifunctional nanocomposite materials have attracted extensive interest because they can integrate multiple functionalities into one single nanosystem and thus endow them with great potential application in various areas (Gao et al., 2009; Liu Z. et al., 2011; Cheng et al., 2013; Jia et al., 2019; Sun et al., 2019; Liao et al., 2020). Among them, nanoparticles (NPs) with magnetism and luminescence have been extensively studied for their applications in biomedicine, for example, targeted enrichment and separation, magnetic targeted drug delivery, magnetic resonance imaging (MRI), biological luminescent probes (Cheng et al., 2016; Chen and Fu, 2018; Ding et al., 2018; Tang et al., 2018; Wang et al., 2018; Fu et al., 2019). Rare-earth doped upconversion luminescent nanomaterials (UCNPs) can emit high-energy photons of different wavelengths in visible or near-infrared (NIR) region by absorbing two or more low-energy photons in NIR region, this makes UCNPs have the prominent advantages over traditional phosphors in low autofluorescence background, shape emission bandwidths, good photochemical stability, and large tissue penetration depth (Xu et al., 2018; Zhu et al., 2018; Zhang et al., 2019). These unique properties proposed UCNPs as a new generation of optical materials in luminescent detections with high sensitivity, high resolution bioimaging, photodynamic therapies (PDT), and so on (Liu J. L. et al., 2011; Liu et al., 2017; Chen S. et al., 2018; Gu et al., 2019; Ma et al., 2019; Tang et al., 2019; Zhu et al., 2019). Magnetic nanoparticles are another kind of attractive materials for biomedicine because they can be fabricated using external magnetic fields. In consequence, the combination of magnetic nanoparticles and UCNPs into one nano-platform is highly pursued in biomedical applications, such as bio-magnetic separation, MRI and optical dual imaging, to improve the targeting efficiency and hyperthermia (Shen et al., 2010; Zhang et al., 2012; Zhong et al., 2012; Chen Z. X. et al., 2018).

Normally, UCNPs could be combined with magnetic materials in three ways. One is the SiO₂-assisted synthetic strategy (Liu et al., 2008). Zhang et al. (2012) synthesized a kind of magnetic upconversion fluoride nanorattles Fe₃O₄@SiO₂@α-NaYF₄/Yb, Er (MUC-F-NR) through an ion-exchange method. The MUC-F-NR consisted a Fe₃O₄ magnetic core, a silica layer, a hollow space, and a NaYF₄/Yb, Er shell. Further studies showed that the nanocomposites have low cytotoxicity, good cell imaging, and excellent tumor therapy efficacy in vivo when treated with an external magnetic field after loading with antitumor drug. The second is the cross-linker anchoring method. Shen et al. (2010) introduced 11-mercaptoundecanoic acid (MUA) or 1,10-decanedicarboxylic acid (DDA) as the crucial crosslinker to immobilize Fe₃O₄ NPs onto the surface of UCNPs, and synthesized the Fe₃O₄/NaYF₄:Yb,Er hetero-NPs. These NPs could be well dispersed in water after ligand ozonolysis treatment, and may also acted as probes for biological imaging. The third is the seed-induced growth method (Zhong et al., 2012; Cheng et al., 2016; Qin et al., 2016). Zhong et al. (2012) first reported a seed-growth procedure to synthesize monodisperse core–shell nanoparticles Fe₃O₄@NaGdF₄:Yb/Er@NaGdF₄:Yb/Er. The Fe₃O₄ core enables the nanocrystals with superparamagnetic property, while the NaGdF₄:Yb/Er outer shell markedly enhances the upconversion emission intensity. In spite that carbon nanomaterials have been extensively studied, the combination of carbon nanomaterials, UCNPs and Fe₃O₄ together to produce nanocomposites has been rarely reported. Zhu et al. (2016) constructed a carbon-coated nanocomposite, NaLuF₄:Yb,Er@NaLuF₄@Carbon (csUCNP@C), which can be used as a real-time monitor of the microscopic temperature in PTT. They found that the microscopic temperatures of the photothermal material upon irradiation were high enough to destroy cancer cells, while the lesions remained low enough to avoid the normal tissue from damage. Liu X. H. et al. (2011) coated a hydrophilic carbon layer on hydrophobic NaGdF₄:Yb, Er nanocrystals to synthesize the core–shell structured NaGdF₄:Yb,Er@Carbon nanocomposites. The prepared nanocomposites were with a uniform size of 25 nm and strong upconversion fluorescence, which can be potentially used as cell-imaging probes. They further developed NaYF₄:Yb,Tm@C@CdS nanoparticles by depositing CdS on carbon-modified NaYF₄:Yb,Tm nanocrystals. The nanocomposites showed good photocatalytic activity due to the efficient energy transfer from NIR light to ultraviolet-visible (UV) light of the NaYF₄:Yb, Tm and the high adsorption ability of the carbon shell (Tou et al., 2016). We have combined Fe₃O₄ and UCNPs together by using a hollow carbon sphere to assemble a cancer theranostic agent. The nanoplatform can realize MRI-UCL dual-mode imaging and PTT for cancer with improved efficiency in vivo (Wang et al., 2019).

Inspired by the previous works mentioned above, we herein provided a kind of multifunctional nanocomposites which combine superparamagnetic Fe₃O₄, UCNPs and a hollow carbon sphere together. The synthesized nanocomposites were fabricated by introducing UCNPs and Fe₃O₄ into a cavity of hollow carbon sphere through a one-pot synthesis method, and the carbon shell wall acts as a protective shell, making it stable and free from the influence of the external environment. Since the carbon layer can convert the absorbed light into heat, the nanocomposites can realize photo thermal (PT), UCL, and MRI tri-mode imaging (Figure 1).

FIGURE 1

Figure 1. Schematic illustration of the synthesized UCNPs/Fe₃O₄@h-C nanocomposites with upconversion luminescence (UCL), magnetic resonance (MR), and photothermal (PT) imagings.

Materials and Methods

Materials

Cyclohexane (AR), anhydrous ethanol (AR), dichloromethane (CH₂Cl₂, AR), acetone (AR), iron (III) chloride hexaydrate (AR), hexamethylenetetramine (HMT, AR) were purchased from Sinopharm Chemical Reagent Co. China. Erbium (III) chloride hexaydrate (ErCl₃·6H₂O, 99.99%), gadolinium (III) chloride hexaydrate (GdCl₃·6H₂O, 99.99%), yttrium (III) chloride hexaydrate (YCl₃·6H₂O, 99.99%), ytterbium (III) chloride hexaydrate (YbCl₃·6H₂O, 99.99%), 1-octadecene (ODE), oleic acid (OA) were purchased from Sigma-Aldrich Co. Ltd. Sodium oleate (CP), n-hexane (AR), methanol (GC), sodium hydroxide (NaOH, GR), sodium oleate (NaOA, AR), ammonium fluoride (NH₄F, GR), 2,4-dihydroxybenzoic acid (DA) were purchased from Shanghai Aladdin Chemistry Co., Ltd (Shanghai, China). All of the chemicals were used without further purification unless specified. A Milli-Q water system (18.2 MΩ•cm, Thermo Fisher) was used to provide the ultrapure water in the experiments.

Synthesis of the Superparamagnetic Nanoparticles Fe₃O₄

The superparamagnetic nanoparticles Fe₃O₄ were synthesized according to a previous reported procedures (Park et al., 2004). Firstly, 5.4 g of FeCl₃·6H₂O and 18.3 g of NaOA were dissolved in a mixed solvent consisting of 40 mL of ethanol, 70 mL of hexane, and 30 mL of deionized water. The mixture was heated to 70°C and kept for 4 hours. After that, the upper organic layer containing iron oleate complex was washed three times with 15 mL of deionized water. After the hexane was evaporated off, the product iron oleate complex was obtained in a waxy solid form. Secondly, 4.5 g of the above synthesized iron oleate complexes and 0.7125 g of OA were dissolved in 32 mL of ODE. The reaction mixture was heated to 320°C and kept at this temperature for 30 min. When the reaction was completed, the solution was cooled to room temperature and 62.5 mL of ethanol was added. The nanocrystals Fe₃O₄ were precipitated and separated by centrifugation and dissolved in n-hexane for further usage.

Synthesis of NaY/GdF₄:Yb,Er

The solutions of YbCl₃ (1 M, 400 μL), ErCl₃ (0.1 M, 400 μL), GdCl₃ (1 M, 500 μL), and YCl₃ (1 M, 1,100 μL) were mixed together and heated to 110°C to evaporate the water. After cooling to room temperature, OA (12.0 mL) and ODE (30.0 mL) were added and the mixture was heated to 150°C to make the solid dissolved completely. Next, 20.0 mL of NaOH (0.2 g) and NH₄F (0.3 g) methanol solution was added and then the reaction solution was heated to 60°C and kept for 30 min. After the evaporating of the methanol under vacuum, the solution was heated to 300°C and maintained for 1 h in an argon (Ar) atmosphere. Then, the solution was added with equal volume of acetone, the products were precipitated and collected by centrifugation and washed three times with the acetone/cyclohexane solution.

Synthesis of NaY/GdF₄:Yb,Er@NaYF₄ (Denoted as UCNPs)

Eight hundred microliter of GdCl₃ (1 M) was heated to 110°C until the water was evaporated completely, then OA (12.0 mL) and ODE (30.0 mL) were added and the mixture was heated to 150°C to make the solid dissolved completely. Next, 5 mL of cyclohexane solution containing the obtained NaY/GdF₄:Yb was added and heated to evaporate the cyclohexane completely. After that, 20.0 mL of NaOH (0.2 g) and NH₄F (0.3 g) methanol solution was added and the temperature was raised to 60°C to evaporate the methanol under vacuum. The next steps were similar to the process of the synthesis of NaY/GdF₄:Yb mentioned above to gain the final product NaY/GdF₄:Yb,Er@NaYF₄, denoted as UCNPs.

Synthesis of Carbon-Based Magnetic Luminescent Nanocomposites (UCNPs/Fe₃O₄@h-C)

Four hundred microliter of hexane solution containing 50 mg of UCNPs and 70 mg of Fe₃O₄ was added into 5 mL of water solution containing 100 mg of NaOA, the mixture was kept under ultrasonic for 10 min to form a water-in-oil emulsion. After the removal of hexane by evaporation at 50°C, the solution was transfer to a 150 mL reactor containing 0.3853 g of DA and 0.0876 g of HMT with 95 mL of deionized water. The mixture was rapidly heated to 160°C and kept for 4 h. The solid was obtained by centrifugation (8,000 rpm, 10 min), washed for 3 times with deionized water, dried at 60°C for 6 h and then heated to 500°C for 2 h under H₂/Ar atmosphere (5%/95%, V/V). The final product is obtained and denoted as UCNPs/Fe₃O₄@h-C.

Characterization

The morphologies of the samples were characterized by transmission electron microscopy (JEM-2010F, JEOL). Fourier transform infrared (FTIR) spectra were measured with Nicolet AVATAR370 FTIR spectroscopy. Upconversion luminescent spectra and luminescence decay curves were acquired by Edinburgh FS5 fluorescent spectroscope with a 0–2 W adjustable 980 nm continuous wave laser. Zeta potentials were measured by Malvern Zetasizer Nano ZSE. The magnetic property of the nanoparticles was performed using a Vibrating Sample Magnetometer (7407, lakeshore) at 298 K. The concentrations of Fe³⁺ were determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The MRI property was measured using a SIMENS 3T MR scanner (MAGNETOM Trio Tim). The r₂ relaxivity was obtained by linear fitting of the 1/T₂ relaxation time (s⁻¹) vs. the Fe³⁺ concentration (mM).

Photothermal Performance Test of UCNPs/Fe₃O₄@h-C Nanocomposites

0.5 mL of UCNPs/Fe₃O₄@h-C of different concentrations (50, 100, 200, and 400 μg mL⁻¹, respectively) dispersed in deionized water, fetal bovine serum (FBS) with different pH value (4, 6, 8), Dulbecco's Modified Eagle's Medium (DMEM), were irradiate under an 808 nm NIR semiconductor laser. The actual output power was precisely calibrated to be 1.5 Wcm⁻² by using an optical power meter. The temperatures were recorded by a thermocouple with an accuracy of 0.1°C every 20 s. All data were acquired from three independent experiments.

The Photothermal Conversion Efficiency Test of UCNPs/Fe₃O₄@h-C Nanocomposites

According to some previous reports (Roper et al., 2007; Zhu et al., 2016; Wei et al., 2017), the photothermal conversion efficiency η of UCNPs/Fe₃O₄@h-C nanocomposites was calculated using the following Equations (1)–(3):

$\begin{array}{ll}\eta =\frac{\text{hS}(T_{max}-T_{surr})-Q_{Dis}}{I(1-10^{-A_{808}})}& (1)\end{array}$

$\begin{array}{ll}{\tau }_s=\frac{m_D{C}_D}{hS}& (2)\end{array}$

$\begin{array}{ll}t=-{\tau }_{s}ln\theta & (3)\end{array}$

In Equation (1), h is heat transfer coefficient, S is the container surface area, T_max is the equilibrium temperature, T_surr is ambient temperature, Q_Dis is heat dissipated from light absorbed by the container itself, which is measured independently containing water without UCNPs/Fe₃O₄@h-C, A₈₀₈ is the absorption intensity of UCNPs/Fe₃O₄@h-C at 808 nm. In Equation (2), τ_s is the sample system time constant, m_D and C_D are the mass and heat capacity of the solvent (water), respectively.

Cell Cytotoxicity Assay in vitro

The cell viabilities were measured with a CCK-8 Kit. Human cervical carcinoma cells (HeLa cells) were used for in vitro cytotoxicity assay of UCNPs/Fe₃O₄@h-C nanocomposites. The cells were seeded in 96-well plates at a density of 8 × 10³ cells per well and incubated overnight in DMEM containing 10% (vol·vol⁻¹) FBS, penicillin (100 μg mL⁻¹), and streptomycin (100 μg mL⁻¹) at 37°C with 5% CO₂, then the culture medium was replaced by fresh DMEM containing different concentrations (50, 100, 150, 200 μg/mL) of UCNPs/Fe₃O₄@h-C for another 24 h. The cell viability of the control group was set as 100% and that of other experimental groups were calculated based on the formula: cell viability = (Abs_450nm of the treated group/Abs_450nm of the control group) ×100%.

Results and Discussion

Synthesis and Characterization of UCNPs/Fe₃O₄@h-C

Firstly, core-shell structured NaY/GdF₄:Yb,Er@NaYF₄ and superparamagnetic nanoparticles Fe₃O₄ were synthesized by a thermal decomposition method. Secondly, acted as the precursor of polymer layer, the interaction between DA and HMT made the solution acidic with a pH of 2.98, under which NaY/GdF₄:Yb,Er@NaYF₄ and Fe₃O₄ nanoparticles were introduced by using NaOA as the surfactant and soft template to transfer oleate-capped NaY/GdF₄:Yb,Er@NaYF₄ and Fe₃O₄ nanoparticles into an aqueous phase. Under a hydrothermal condition, HMT is decomposed into NH₃ and HCHO to produce cavity structure. Meanwhile, during the heating process, NaOA emulsion droplets are heated and expanded, making the cavity volume grow larger. Finally, the temperature of the mixture was raised to 500°C and kept for 2 h under the atmosphere of Ar (95%) and H₂ (5%) to generate the carbon shell from DA reduction (Sun et al., 2013). As shown in Figure 2A, the Fe₃O₄ has monodisperse morphology, and the particle size is about 12 nm calculated from the TEM images using ImageJ software (Figure S1a, Supplementary Information). In order to improve the luminescence intensity of NaY/GdF₄:Yb,Er, a shell NaYF₄ was coated on its outer layer to obtain the core-shell structure upconversion nanoparticles NaY/GdF₄:Yb,Er@NaYF₄ (denoted as UCNPs), as shown in Figure 2B. UCNPs had uniform morphology, good dispersion, and the particle sizes ranging from 18 to 21 nm (Figure S1b, Supplementary Information). The carbon-based multifunctional nanocomposites were finally obtained by means of the hollow carbon sphere to make Fe₃O₄ and UCNPs locate in the hollow cavity at the same time. As shown in Figure 2C, the synthesized UCNPs/Fe₃O₄@h-C nanocomposites have a hollow structure with a huge cavity about 160 nm in diameter, and the carbon layer was about 60–70 nm in thickness. The total size of the UCNPs/Fe₃O₄@h-C is calculated to be 280–300 nm Figure S1c, Supplementary Information). Moreover, we found that UCNPs and Fe₃O₄ existed simultaneously in the hollow cavity of the nanocomposites, UCNPs with a relatively large particle size (denoted by the red arrow) and Fe₃O₄ with a relatively small particle size (denoted by the green arrow) were observed at the same time. Compared with NaY/GdF₄:Yb,Er@NaYF₄ NPs, the particle size of UCNPs decreased when it entered into the hollow cavity of carbon sphere, which may be caused by OA-induced dissolution of the core-shell nanocrystals (Liu et al., 2016; Tang et al., 2019). The zeta potentials of NaY/GdF₄:Yb,Er, UCNPs, and UCNPs/Fe₃O₄@h-C are −5.2 ± 1.13 mV, −4.0 ± 0.62 mV, and −7.5 ± 1.44 mV, respectively (Figure S2, Supplementary Information). Since the nanocomposites contain carbon materials, upconversion luminescence materials and magnetic materials, it is preliminarily believed that this nanosystem can realize photothermal, UCL and MRI trimode imaging simultaneously.

FIGURE 2

Figure 2. Transmission electron microscopy (TEM) images of Fe₃O₄ (A), UCNPs (B), and UCNPs/Fe₃O₄@h-C nanocomposites (C). The red arrow indicates the UCNPs, and he green arrow indicates the Fe₃O₄.

The FTIR spectra of Fe₃O₄, UCNPs and UCNPs/Fe₃O₄@h-C are exhibited in Figures 3A–C, respectively. In Figure 3A, the peaks at 2,917 and 2,852 cm⁻¹ correspond to the asymmetric and symmetric stretching vibrations of methylene (-CH₂-) in the oleic acid chain, the peaks at 1,535 cm⁻¹ correspond to the vibration of the unsaturated carbon-oxygen double bond (-C=O-) in the oleic acid, the peaks at 1,445 cm⁻¹ correspond to the vibration of the submethyl (-CH), and the peaks at 594 cm⁻¹ correspond to the Fe-O bond in Fe₃O₄. In Figure 3B, the asymmetric and symmetric stretching vibrations of methylene (-CH₂-) of the UCNPs are at 2,915 and 2,860 cm⁻¹, respectively, the vibration of the unsaturated carbon-oxygen double bond (-C=O-) and the submethyl (-CH) were corresponding to the peaks at 1,567 and 1,457 cm⁻¹, respectively. In Figure 3C, similar peaks at 2,917, 2,850, 1,556, 1,454 cm⁻¹ were observed, which assigned to the asymmetric and symmetric stretching vibrations of methylene (-CH₂-) and the vibration of the unsaturated carbon-oxygen double bond (-C=O-). These peaks are attributed to the present of -CH₂-CH₂- and carbon-oxygen groups of hollow carbon nanospheres, as well as some contributions from the surface groups of UCNPs and Fe₃O₄ nanoparticles. Meanwhile, the peak at 602 cm⁻¹ corresponding to the Fe-O bond suggested the existence of Fe₃O₄. The peak at 3,411 cm⁻¹ corresponding to the stretching vibration of hydroxyl (OH) may due to the water molecules adsorbed and the hydroxyl on the surface of carbon spheres.

FIGURE 3

Figure 3. Fourier transform infrared (FTIR) spectra of Fe₃O₄ (a), UCNPs (b), and UCNPs/Fe₃O₄@h-C nanocomposites (c).

Magnetic and UCL Properties of UCNPs/Fe₃O₄@h-C

Due to the presence of Fe₃O₄ in the hollow nanocavity, the nanocomposites UCNPs/Fe₃O₄@h-C are expected to have good magnetic properties. The hysteresis loops of Fe₃O₄ (red) and UCNPs/Fe₃O₄@h-C (blue) are shown in Figure 4. Both Fe₃O₄ NPs and the UCNPs/Fe₃O₄@h-C nanocomposites showed superparamagnetic properties because no hysteresis was observed in the figures. The saturation magnetization of Fe₃O₄ is 26.68 emu/g. When Fe₃O₄ NPs and UCNPs were encapsulated into the hollow carbon cavity together, the saturation magnetization was markedly reduced to 0.87 emu/g, which was a little smaller than that of the samples (1.28 emu/g) prepared by Zhang et al. (2012). This may mainly because of the weight contribution from nonmagnetic NaY/GdF₄:Yb,Er@NaYF₄ materials and the hollow carbon materials. The magnetic separation ability of UCNPs/Fe₃O₄@h-C was further studied. Under the action of external magnetic field on one side of glass tube, these black nanoparticles are attracted by magnets in a short time, which proves the existence of magnetic Fe₃O₄ (Inset photos in Figure 4). Therefore, although the saturation magnetization value of UCNPs/Fe₃O₄@h-C is decreased, it still showed effective magnetic separation capability. These results demonstrated that the nanocomposites are expected for bioapplications in magnetic enrichment or separation.

FIGURE 4

Figure 4. Magnetic hysteresis loops of Fe₃O₄ and UCNPs/Fe₃O₄@h-C nanocomposites at room temperature. Inset shows the digital photographs of UCNPs/Fe₃O₄@h-C nanocomposites without (A) or with (B) a commercial permanent magnet.

The upconversion luminescence spectra of NaY/GdF₄:Yb,Er@NaYF₄ and UCNPs/Fe₃O₄@h-C nanocomposites were measured and shown in Figure 5. When excited by a 980 nm laser, both NaY/GdF₄:Yb,Er@NaYF₄ and UCNPs/Fe₃O₄@h-C exhibited three independent characteristic peaks located at about 524, 545 and 654 nm, they were assigned to the ²H_11/2→⁴I_15/2, ⁴S_3/2→⁴I_15/2 and ⁴F_9/2→⁴I_15/2 transitions of Er³⁺ ions, respectively. The characteristic emission peaks of UCNPs/Fe₃O₄@h-C nanoparticles are similar to those of NaY/GdF₄:Yb,Er@NaYF₄, suggesting that the doping of Fe₃O₄ NPs does not basically impeded the luminescent performance of NaY/GdF₄:Yb,Er@NaYF₄ despite some partial quenching caused by the black Fe₄O₃ and the hollow carbon layer. The visualized presentation of UCL can also be tested by the photo taken from a digital camera, as shown in inset of Figure 5, visible green luminescence can be obviously seen when UCNPs/Fe₃O₄@h-C nanocomposites is irradiated with 980 nm excitation light. The luminescence decay curves of Er³⁺: ⁴S_3/2→⁴I_15/2 transition in NaY/GdF₄:Yb,Er, NaY/GdF₄:Yb,Er@NaYF₄, and UCNPs/Fe₃O₄@h-C under 980 nm excitation was shown in Figure S3, and the lifetimes were calculated as 0.04, 0.46, and 0.02 ms for NaY/GdF₄:Yb,Er, NaY/GdF₄:Yb,Er@NaYF₄, and UCNPs/Fe₃O₄@h-C, respectively. These results indicate that the obtained nanocomposites can retain the upconversion luminescence properties from UCNPs, which can be exploited in optical imaging in vivo.

FIGURE 5

Figure 5. Upconversion luminescence spectra of UCNPs and UCNPs/Fe₃O₄@h-C nanocomposites. Inset shows the digital photograph of UCNPs/Fe₃O₄@h-C nanocomposites under 980 nm laser irradiation.

In vitro Photothermal Effect and MR Imaging of UCNPs/Fe₃O₄@h-C

It is well known that carbon materials have good heat-absorbing properties and can convert the absorbed NIR light into heat efficiently. Herein, the temperature changes of UCNPs/Fe₃O₄@h-C solution at different concentrations under continuous laser irradiation at 808 nm were measured to evaluate the photothermal conversion of the nanocomposites. As indicated in Figure 6, the temperature of the solution increased by 12.2, 15.2, 25.7, and 34.6°C after 7 min of illumination when the concentrations of the sample were 50, 100, 200, and 400 μg/mL, respectively. Compared with the control group of pure water, the temperature only increased by 8°C under the same conditions (Figure 6A). The photothermal conversions of the nanocomposites in different solutions were also evaluated. As shown in Figure S4 in Supplementary Information, similar trends of temperature changes were observed when UCNPs/Fe₃O₄@h-C dispersed in FBS with different pH value (4, 6, 8), or cell culture medium DMEM, which indicated that the solvents had little influence on the photothermal effect of UCNPs/Fe₃O₄@h-C composite. We further tested the thermal stability of UCNPs/Fe₃O₄@h-C nanocomposites. The sample (200 μg/mL) was irradiated with 808 nm laser for 7 min and then cooled naturally to room temperature. As shown in Figure 6B, the temperature changes of the three heating and cooling experiments are similar, indicating that the samples have good thermal stability. Furthermore, the infrared thermal images of UCNPs/Fe₃O₄@h-C with different concentrations at the time points of 1.5, 3, 4.5, 6, and 7.5 min were recorded. As shown in Figure 6C, the temperatures of the UCNPs/Fe₃O₄@h-C nanocomposites increased rapidly with the increase of concentration from 0 to 400 μg/mL. In addition, time constant of heat transfer from this system is determined to be τ_s = 181.6 s, and the photothermal conversion efficiency (η) was calculated to be 24.07% from the obtained data (Figures 7A,B). Thus, the nanocomposites with a good photothermal conversion effect and good photothermal stability may become a promising photothermal imaging and photothermal treatment agent for tumors.

FIGURE 6

Figure 6. (A) The temperature changes of water, UCNPs/Fe₃O₄@h-C nanocomposites at different concentrations (50, 100, 200, 400 μg/mL, respectively) as a function of time under 808 nm laser irradiation at a power density of 1.5 W cm⁻². (B) The temperature changes of UCNPs/Fe₃O₄@h-C nanocomposites during the three photothermal cycles. (C) Infrared thermal images of the different concentrations of UCNPs/Fe₃O₄@h-C nanocomposites at the time points of 1.5, 3, 4.5, 6, and 7.5 min under 808 nm laser irradiation (1.5 W cm⁻²).

FIGURE 7

Figure 7. (A) The temperature changes of UCNPs/Fe₃O₄@h-C nanocomposites (200 μg mL⁻¹) after 808 nm laser irradiation (1.5 Wcm⁻²) for 7 min, following the laser being turned off. (B) Time constant for heat transfer from the system calculated to be τ_s = 181.6 s by using the linear time data from the cooling period (after 420 s) vs. the negative natural logarithm of the driving force temperature, which is obtained from the cooling profile in Figure 6A.

Fe₃O₄ is well known as a contrast agent for MRI technique, which widely applied for noninvasive biological tissues imaging with high organ resolution. Figure 8A shows relaxation rate r₂ (1/T₂) plotted against the different Fe³⁺ concentrations of UCNPs/Fe₃O₄@h-C nanocomposites. The r₂ value of UCNPs/Fe₃O₄@h-C is calculated by the linear curve with a slope of 57.7 mM⁻¹S⁻¹, indicated that the UCNPs/Fe₃O₄@h-C nanocomposites have potential applications in MRI as T₂-weighted contrast agents. As shown in Figure 8B, the signal intensities decrease gradually with the increase of Fe³⁺ concentration, which can be seen vividly in a color mapped images.

FIGURE 8

Figure 8. (A) Relaxation rate r₂ (1/T₂) plotted against the different Fe³⁺ concentrations of UCNPs/Fe₃O₄@h-C nanocomposites. (B) T₂-weighted and color-mapped magnetic resonance (MR) images for various Fe³⁺ concentrations of UCNPs/Fe₃O₄@h-C nanocomposites.

In vitro Cytotoxicity Assay of UCNPs/Fe₃O₄@h-C

The cytotoxicity of UCNPs/Fe₃O₄@h-C nanocomposites in HeLa cells was evaluated by using CCK-8 assay. As shown in Figure S5 in Supplementary Information, the cell viabilities were over 90% at all tested concentrations from 50 to 200 μg/mL, suggesting that UCNPs/Fe₃O₄@h-C has excellent biocompatibility.

Conclusions

In conclusion, monodisperse, multifunctional UCNPs/Fe₃O₄@h-C nanocomposites have been successfully prepared. The NaY/GdF₄:Yb,Er@NaYF₄ UCNPs of the nanocomposites can convert the absorbed 980 nm NIR light into visible luminescence for UCL imaging, the carbon layer of the nanocomposites can convert the absorbed 808 nm NIR light into heat to realize photothermal imaging. Furthermore, the r₂ value of UCNPs/Fe₃O₄@h-C (57.7 mM⁻¹S⁻¹) indicates the nanocomposites could be used as potential T₂-weighted MRI contrast agents. To the best of our knowledge, this is the first report to load UCNPs and magnetic nanoparticle simultaneously in the cavity of the hollow carbon cavity to achieve upconversion luminescence, magnetic resonance, and photothermal imaging together.

Data Availability Statement

The datasets generated for this study are available on request to the corresponding author.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given their approval of the final version of the manuscript.

Funding

We acknowledge the financial support from Weifang Science and Technology Development Plan Project (No. 2018GX108), the Open Experimental Project of Shandong Peninsula Engineering Research Center of Comprehensive Brine Utilization (No. 2018LS017), National Natural Science Foundation of China (No. 31671011), and Innovative Research Team of High-Level Local Universities in Shanghai.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2020.00611/full#supplementary-material

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