In situ crystal growth of gold nanocrystals on upconversion nanoparticles for synergistic chemo-photothermal therapy

Ruoyan Wei a, Wensong Xi b, Haifang Wang b, Jinliang Liu a, Torsten Mayr c, Liyi Shi a and Lining Sun *a
aResearch Center of Nano Science and Technology, and School of Material Science and Engineering, Shanghai University, Shanghai 200444, China. E-mail:; Tel: +86-21-66137153
bInstitute of Nanochemistry and Nanobiology, Shanghai University, Shanghai 200444, China
cApplied Sensors, Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, 8010 Graz, Austria

Received 31st March 2017 , Accepted 13th June 2017

First published on 14th June 2017

A multifunctional cancer therapy nanocomposite was proposed and synthesized by linking the pH-responsive SH-PEG-DOX prodrug onto gold nanocrystals that were grown in situ on the surface of upconversion nanoparticles (UCNPs). In the structure of the SH-PEG-DOX prodrug, a hydrazone bond was utilized for subsequent pH-responsive drug release in the intracellular acidic microenvironment of cancer cells. This innovative assembly method is facile and mild, and can be used to obtain nanocomposites of UCNPs and gold, which show excellent photostability and biocompatibility. The final UCNPs@Au-DOX nanocomposites offer efficient treatment effects in vitro under irradiation with an 808 nm laser due to the synergistic effect of chemotherapy and photothermal therapy. In addition, the UCNPs@Au-DOX nanocomposites show excellent intracellular locating ability via upconversion luminescence (UCL) imaging with Er3+ ions and magnetic resonance imaging (MRI) with Gd3+ ions, indicating that they have potential as a visual tracking agent in cancer treatment. Therefore, the presented bioimaging-guided multifunctional synergistic therapy nanocomposites are promising tools for imaging-guided cancer therapy.

1. Introduction

Up to now, nanomaterials for chemical or physical therapy guided by near infrared (NIR) light imaging have played an increasingly important role in the field of visual cancer therapy. As a matter of fact, both chemical and physical cancer therapies have their own limitations. Bioimaging-guided cancer therapy based on nanocomposites has attracted significant attention in recent years due to its higher therapeutic efficiency and reduced side effects.1–5 Traditionally, non-targeted chemotherapy with anti-cancer drugs results in low therapeutic efficacy and relatively high cytotoxicity to the whole system. Recently, stimuli-responsive drug release of nanomaterials, especially pH-responsive prodrugs, has been widely explored in biological fields, which benefits from the intracellular acidic microenvironment of cancer cells.6–8 However, the relatively low drug loading capacity, the rapid drug release speed, and drug resistance primarily reduce their effectiveness. Compared to chemotherapy, photothermal (physical) therapy (PTT) has emerged as a noninvasive therapeutic technique with controllable operation, high selectivity, and low systemic toxicity.9–11 Thus, the synergistic effect of chemotherapy and physical therapy is expected to improve the effect of cancer treatment. Some widely used photothermal agents present photothermal conversion effects, including gold nanoparticles,12,13 platinum (Pt) nanoparticles,14 WO2.9 nanorods,15 Cu7.2S4 nanocrystals,16 MoS2 nanosheets,17 Bi2S3 nanorods,5 carbon nanomaterials,18,19 polymeric nanoparticles,20,21etc. Among all of these photothermal materials, gold nanocrystals have made a significant contribution to the fundamental research of how nanomaterials behave both in vitro and in vivo. Up to now, gold nanomaterials have not only enabled various diagnostic techniques, such as immunoassays,22 bioimaging,23,24 and biosensors,25 but have also shown prospective potential in targeted therapeutics, especially photothermal therapy (due to their excellent photothermal conversion effect),26 and cancer treatment.27,28 For example, under irradiation of a NIR laser, the temperature of gold nanocrystals increases intensely in cancer cells, which are more sensitive to heat in comparison to normal cells, and thus they can be effectively killed at high temperature. Therefore, gold nanocrystals can be successfully used in photothermal therapy for treatment of tumors.29

In addition, to track and locate the nanocomposites for synergistic therapy, a visual tracking agent is required. Rare earth-doped upconversion nanoparticles (UCNPs) are capable of emitting UV, visible and NIR light under excitation with continuous wave (CW) NIR radiation.30 Compared to UV and visible light, NIR radiation can penetrate deeper into tissues and causes less damage to organisms with low autofluorescence background, thus making UCNPs outstanding visual tracking agents.31 Most recently, there have been a variety of reports demonstrating that UCNPs can act as excellent visual nanocarriers for drug and photothermal agents.32,33 Up to now, most nanocomposites based on UCNPs and gold nanocrystals have been synthesized through specific chemical or physical combination after they have been prepared from specific methods in advance separately. However, these processes are generally complicated and time consuming. For example, Yang's group prepared amino-functionalized UCNPs@MS first and then absorbed electronegative Au25 nanoparticles, which were successfully used as an 808 nm NIR light-induced cancer therapy platform.32 Recently, Yang et al. reported an in situ growth strategy to combine UCNPs with ultrasmall CuS particles for photothermal theranostics, which could be spread to other integration processes.34 In this report we present nanocomposites for photothermal therapy, drug release, upconversion luminescence imaging, and magnetic resonance imaging. Photothermal functionality is obtained using gold nanocrystals, which are directly grown in situ on the surface of UCNPs, in which the gold nanocrystals serve as photothermal conversion agents under excitation with an 808 nm laser. The modified UCNPs are used as visual tracking agents and for upconversion luminescence and magnetic resonance imaging. In addition, a pH-responsive prodrug is synthesized and coupled to the gold–UCNP nanocomposites, serving as a chemotherapeutic agent and endowing the synergistic effect of chemotherapy and photothermal therapy to the final nanomaterials.

Herein, we demonstrate a facile method to synthesize nanocomposites based on direct in situ growth of gold nanocrystals on the surface of UCNPs. The obtained cit-UCNPs@Au nanoparticles show excellent photostability and biocompatibility. In addition, a new chemotherapy prodrug, SH-PEG-DOX, was synthesized and conjugated to the cit-UCNPs@Au to enhance the cancer treatment effect of chemotherapy and photothermal therapy. The final UCNPs@Au-DOX nanocomposites were investigated in HeLa cells upon irradiation at 808 nm using a series of CCK-8 cytotoxicity assays, showing a very low cell viability and an excellent intracellular synergistic effect of chemotherapy and photothermal therapy. Their successful application in intracellular upconversion luminescence (UCL) and magnetic resonance imaging (MRI) indicates that the UCNPs@Au-DOX nanocomposites are potential tools for multimodal bioimaging-guided treatment.

2. Materials and methods

2.1. Materials

All reagents and chemicals were obtained from commercial sources and used without further purification. Ultrapure water was used throughout. YCl3·6H2O (99.9%), YbCl3·6H2O (99.9%), ErCl3·6H2O (99.9%), GdCl3·6H2O (99.9%), gold(III) chloride trihydrate (≥49.0%), 1-octadecane (ODE, 90%), oleic acid (OA, 90%), Dulbecco's modified Eagle's medium (DMEM), and fetal bovine serum (FBS) were purchased from Sigma-Aldrich Co. Ltd. Sodium hydroxide (NaOH, 96%), ammonium fluoride (NH4F, 98%), methanol (CH3OH, 99.5%), doxorubicin hydrochloride (DOX·HCl), diethylene glycol (DEG) and N,N-dimethylformamide (DMF) were obtained from Aladdin Company. Cyclohexane, acetone, phosphoric acid (H3PO4) and sodium borohydride (NaBH4) were of analytical grade and purchased from Sinopharm Chemical Reagent Co. Ltd. SH-PEG-CONHNH2 (SH-PEG-HZ, MW = 5000, 90%) was purchased from Shanghai Ponsure Biotech, Inc. The WST-8 cell counting kit (CCK-8) was purchased from Dojindo Molecular Technologies Inc. 96-well plates were obtained from Greiner Bio One GmbH. D-hanks solution was obtained from Gibco Inc. The live/dead cell staining kit was obtained from Invitrogen.

2.2. Synthesis of the SH-PEG-DOX prodrug

The SH-PEG-DOX prodrug was synthesized based on the reported method with some modification.35 SH-PEG-HZ (0.75 g, 0.15 mmol) and DOX·HCl (0.029 g, 0.05 mmol) were added into 10 mL of a DMF solution with stirring, and 10 μL of H3PO4 was introduced as a catalyst. The reaction mixture was stirred for another 48 h at room temperature. The resulting mixture was dialyzed against PBS buffer solution (pH = 8.0) through a dialysis membrane (MWCO of 3500) until the PBS solution became colorless. Finally, the SH-PEG-DOX powder was freeze-dried under vacuum at ambient conditions for 48 h. 1H NMR (500 MHz, D2O) δ (ppm): 7.48 (t, CH of DOX), 7.20 (m, CH of DOX), 5.35 (s, OH of DOX), 3.68 (m, CH2 of PEG).

2.3. Synthesis of citric acid-modified UCNPs (denoted as cit-UCNPs)

NaYF4:Yb,Er@NaGdF4 (abbreviated as UCNPs) was synthesized according to our previously reported solvothermal methods.36,37 Then, citrate capped UCNPs were obtained through a ligand exchange process. Citric acid (1.0 g) was added into 30.0 mL of DEG solution, and then heated to 110 °C for 30 min under argon. After the solution was cooled down to 60 °C, 25 mg of UCNPs dispersed in chloroform and toluene (3/2 v/v) solution (10 mL) was injected into the above mixed solution, and the mixture was heated to 130 °C for 1 h to evaporate the chloroform and toluene. Finally, the system was further heated to 175 °C for 2 h until the solution became yellow under argon. The resulting solution was cooled down to room temperature, and the products were collected by centrifugation (15[thin space (1/6-em)]000 rpm, 15 min). The precipitates were washed three times with ethanol and ultrapure water (1/1 v/v) solution (20 mL), and the final citric acid-capped UCNPs (abbreviated as cit-UCNPs) were re-dispersed in 5 mL of ultrapure water.

2.4. In situ crystal growth of gold nanocrystals on the surface of the cit-UCNPs (denoted as cit-UCNPs@Au)

100 μL of 1% HAuCl4·3H2O aqueous solution and 1 mL of cit-UCNPs were mixed in a 10 mL round-bottom flask and vigorously stirred for 100 min at room temperature. Afterwards, 500 μL of NaBH4 (50 mM) aqueous solution in ice-cold water was rapidly injected into the above mixture, which was further stirred for another 2 h until the solution became deep red. The reaction mixture was centrifuged (10[thin space (1/6-em)]000 rpm, 15 min) to collect the products. Then, the products were washed with 10 mL of ultrapure water several times, and the cit-UCNPs@Au nanoparticles were dispersed in 2 mL of ultrapure water and stored at 4 °C until further use.

2.5. Linking SH-PEG-DOX on the cit-UCNPs@Au nanoparticles (denoted as UCNPs@Au-DOX)

First, 2 mg of SH-PEG-DOX was dissolved in 5 mL of ultrapure water. Then, 2 mL of a cit-UCNPs@Au aqueous solution was added dropwise into the above solution, and the solution was stirred overnight in the dark at room temperature. The resulting solution was centrifuged (10[thin space (1/6-em)]000 rpm, 15 min) to collect the final nanocomposites (UCNPs@Au-DOX), and the nanocomposites were washed with ultrapure water twice and re-dispersed in ultrapure water.

2.6. Characterization

Different nanoparticles including UCNPs, cit-UCNPs, cit-UCNPs@Au and UCNPs@Au-DOX dispersed in cyclohexane or ultrapure water were carefully dropped onto a 300-mesh copper grid coated with a lacy carbon film, and imaged on a JEM-2100F low-to-high resolution transmission electron microscope (TEM) operated at 120 kV. Fourier transform infrared spectra (FT-IR) were obtained on a Thermo Nicolet 6700 spectrometer with KBr pellets in the spectral range of 4000 to 400 cm−1. The crystal phases of the nanoparticles were identified by powder X-ray diffraction (XRD) measurements on an 18 kW D/MAX2500V+PC diffractometer at a scanning rate of 8° min−1 with 2θ from 10° to 90° (Cu Kα radiation, 60 kV, 80 mA). The concentrations of Gd3+ and Au3+ were determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The upconversion luminescence spectra were obtained using an Edinburgh LFS-920 fluorescence spectrometer with a 0–2 W adjustable continuous wave laser (980 nm, Connect Fiber Optics, China). UV-Vis spectra were recorded on a Shimadzu UV-2500PC ultraviolet-visible spectrometer. Thermogravimetric (TG) curves were measured using an STA409PC (NETZSCH-Gerätebau GmbH). The Z-potential (zeta) and dynamic light scattering (DLS) were measured on a Potentiometric Analyzer Zetasizer 3000HS using PCS analysis software (Malvern Instruments Corporation). The wavelength was set as 633 nm during the whole experiment. The 808 nm laser was equipped with an external adjustable power source (0–2 W cm−2) and a 5 mm diameter laser module (Xi'an Sapling Laser Techine Institute, China). In the cell viability test, the optical density (OD) of each well at 450 nm was recorded on a microplate reader (Thermo, Varioskan Flash, USA). The live/dead staining was observed using a fluorescence microscope (DMI3000, Leica, Germany) under excitation at 495 and 528 nm, respectively. Cellular UCL fluorescence images were obtained digitally on a Nikon multiple charge-coupled device (CCD) using a confocal laser scanning microscope (CLSM).

2.7. In vitro drug release of UCNPs@Au-DOX

The in vitro DOX release investigations were performed by placing UCNPs@Au-DOX at a concentration of 1 mg mL−1 in a dialysis bag (3500 MW) into 4 mL of PBS buffer solution (pH = 7.4, 6.0, or 4.5) at 37 °C under continuous stirring. At specified time intervals of 2, 4, 6, 8, 10, 12, 24, and 48 h, 2 mL of the original PBS was replaced with fresh PBS and the as-obtained original PBS was analyzed using a UV-Vis spectrometer at 480 nm in order to determine the amount of DOX released.

2.8. In vitro 808 nm laser-induced photothermal (PTT) effect of UCNPs@Au-DOX

For measuring the PTT effect of the UCNPs@Au-DOX nanocomposites, an 808 nm NIR semiconductor laser device with a 5 mm diameter laser module was transported through a glass bottle containing a UCNPs@Au-DOX aqueous solution (0.5 mL) with different concentrations (50, 100, 200, and 400 μg mL−1). The light source was externally adjustable, and the actual output power was precisely calibrated using an optical power meter to be 1.5 W cm−2. A thermocouple with an accuracy of 0.1 °C was inserted into the aqueous dispersion of UCNPs@Au-DOX perpendicular to the path of the laser. The temperature was recorded every 20 s. All data were acquired from three independent experiments to ensure the reliability of the data.

2.9. Cell viability of HeLa cells after exposure to DOX, cit-UCNP@Au, and UCNPs@Au-DOX under irradiation with an 808 nm laser

Human cervix carcinoma cells, HeLa cells, were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The HeLa cells were maintained in high glucose DMEM (4.5 g L−1 glucose) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in an atmosphere of 5% CO2/95% air.

The WST-8 cell counting kit (CCK-8, Dojindo Molecular Technologies Inc., Kumamoto, Japan) was used to investigate the in vitro toxicity of DOX, cit-UCNPs@Au, and UCNPs@Au-DOX. HeLa cells (5000 per well) in 100 μL of culture medium were plated in the wells of 96-well plates and grown overnight. Then, the medium was removed and 200 μL of fresh culture medium containing cit-UCNPs@Au or UCNPs@Au-DOX nanocomposites at different concentrations (0, 50, 100, and 200 μg mL−1), or free DOX at the corresponding concentrations (0, 3.75, 7.5, and 15 μg mL−1) was introduced to the cells for 4 h at 37 °C under 5% CO2/95% air. For comparison, two wells of each plate were selected that were far away from each other (to avoid the influence of the temperature increasing during the photothermal experiment) and the same sample was introduced into them. One well was irradiated with an 808 nm laser for 24 min (8 min break after each 8 min irradiation) and the other was not irradiated. HeLa cells alone served as the control group. Then, all wells of the plates were further incubated for another 24 h. After removing the culture medium, CCK-8 solution (100 μL, containing 10% CCK-8) was added to each well and incubated for 1.5 h. The viability of the HeLa cells was measured by a microplate reader at the wavelength of 450 nm. The cell viability (% of control) is expressed as the percentage of (ODtest − ODblank)/(ODcontrol − ODblank), where ODtest is the OD of the cells exposed to free DOX, cit-UCNPs@Au, or UCNPs@Au-DOX, ODcontrol is the OD of the control, and ODblank is the OD of the well without cells. All of the tests were independently performed six times.

2.10. Live/dead staining of HeLa cells after exposure to cit-UCNPs@Au and UCNPs@Au-DOX

A live/dead cell assay was performed by staining the live/dead cells with calcein acetoxymethyl (calcein AM) or propidium iodide (PI) to image the different sample-treated cells. The mixture of calcein AM and ethidium homodimer-1 in the kit can differentiate live (green, Ex: 495 nm; Em: 515 nm) cells from dead (red, Ex: 528 nm; Em: 617 nm) cells. Cells were treated with different samples as described above. After 24 h of incubation, the culture medium was removed and the cells were washed with D-hanks. Then, the mixture of dyes dissolved in D-hanks was added to wells and the cells were incubated for another 30 min at room temperature in the dark. Then, the excess dye was removed and the cells were washed carefully with D-hanks. Finally, 100 μL of D-hanks solution was added, and the cells were investigated under a fluorescence microscope.

2.11. Cellular internalization and localization in HeLa cells

Cellular internalization and localization was performed using a confocal laser scanning microscope (CLSM) with a 980 nm laser (Connet Fiber Optics, China) with a focused power of ∼500 mW as an additional excitation source. Before the experiments, HeLa cells were plated in Petri dishes and grown overnight, and then the medium was removed and 2 mL of fresh culture medium containing 200 μg mL−1 UCNPs@Au-DOX nanocomposites was added, and the HeLa cells were further incubated for 2 and 4 h at 37 °C under 5% CO2/95% air. CLSM cell imaging was then carried out after washing the cells with DMEM five times. The imaging of DOX was carried out under 488 nm laser excitation, and the emission light was collected at 550–700 nm, while the UCL emissions of green and red luminescence from the UCNPs were collected at 500–600 nm and 600–700 nm, respectively.

2.12. Longitudinal relaxation time T1 and relaxivity r1 measurement

The longitudinal relaxation time T1 and relaxivity r1 of the UCNPs@Au-DOX were measured on a magnetic resonance imaging (MRI) instrument with a 3.0 T magnetic field. A series of different concentrations of UCNPs@Au-DOX nanoparticles (containing 0–1.55 mM Gd3+) dispersed in water were placed in 2 mL plastic centrifuge tubes for the T1 measurements. The resulting values of 1/T1 were plotted with the concentrations of Gd3+, and the longitudinal relaxivity r1 was deduced from the slope of the fitted regression line.

3. Results and discussion

3.1. Synthesis and characterization of the UCNPs@Au-DOX nanocomposites

Rare earth upconversion nanoparticles with a core–shell structure (NaYF4:Yb,Er@NaGdF4, UCNPs) were synthesized, and an NaGdF4 shell with similar lattice constants compared with NaYF4 was used to enhance the intensity of the UCL of the core nanoparticles and provide the UCNPs with potential MRI ability, as shown in Scheme 1. Generally, citric acid is a common stabilizing agent and reducing agent for the synthesis of gold nanocrystals.38 The UCNPs were assembled with citric acid based on ligand exchange according to our modified procedure.39,40 After ligand exchange, the resulting cit-UCNPs were sufficiently hydrophilic due to the multiple carboxyl groups on the surface of the UCNPs. In the next step, the citric acid on the cit-UCNPs severed as both a stabilizing agent of the UCNPs and an assistant reducing agent of HAuCl4 in the in situ crystal growth process of gold nanocrystals on the surface of the cit-UCNPs, in which NaBH4 served as the co-reducer. This strategy is free from the use of any surfactant or template, and reveals that the direct reduction of HAuCl4 on the surface of cit-UCNPs is highly feasible to obtain photothermal agents. In addition, a chemotherapy prodrug of SH-PEG-DOX was synthesized and conjugated to the cit-UCNPs@Au nanoparticles to enhance the chemotherapy and photothermal therapy effects for cancer treatment, attributed to the strong bond between –SH and the gold nanocrystals. Up to now, the majority of gold nanoparticles have been modified through the creation of a strong Au–S bond, where Au can easily complex with the –SH groups.41,42 In the structure of SH-PEG-DOX, a hydrazone bond was introduced, which is beneficial for pH-responsive drug release in the acidic microenvironment of cancer cells.
image file: c7nr02280h-s1.tif
Scheme 1 Schematic illustration of the synthesis, upconversion luminescence (UCL)/magnetic resonance (MR) imaging, intracellular chemotherapy, and photothermal therapy (PTT) of UCNPs@Au-DOX nanocomposites.

As can be seen in Fig. 1a and b, after modification with citric acid, the morphology and size of the UCNPs remained relatively unchanged, and the cit-UCNPs showed good mono-dispersity with a size of approximately 30 nm. From Fig. 1c, it can be clearly observed that gold nanocrystals were grown on the surface of the cit-UCNPs uniformly. The crystal growth of the gold nanocrystals on the cit-UCNPs has no obvious impact on the morphology of the cit-UCNPs. The HRTEM image of cit-UCNPs@Au clearly shows the formation of uniformly spherical gold nanoparticles with a size of ∼5 nm on the UCNPs (Fig. 1d). Furthermore, both the gold nanocrystals and the UCNPs maintain their great crystal shape and show distinct lattice fringes (Fig. S1). It is noteworthy that there is stacking of the lattice fringes between gold and the UCNPs, indicating that the gold nanocrystals were grown on the surface of the UCNPs, rather than simple physical combination. The presence of the elements Au, Y, Yb, Gd, Er, Na, O, and F from the energy dispersive X-ray (EDX) spectrum and elemental mapping images of the cit-UCNPs@Au in Fig. S2 suggests that the cit-UCNPs@Au nanoparticles were successfully synthesized. As shown in Fig. 1e, the scanning transmission electron microscopy-high angle annular dark-field (STEM-HAADF) image of the final UCNPs@Au-DOX nanocomposites demonstrates that after linking with SH-PEG-DOX, there is no obvious aggregation. In addition, the dynamic light scattering (DLS) measurements showed that the intensity-average diameters of the cit-UCNPs, cit-UCNPs@Au, and UCNPs@Au-DOX nanocomposites were 52.1 nm, 74.8 nm, and 161.1 nm, respectively (Fig. S3). The zeta potentials of the cit-UCNPs, cit-UCNPs@Au, SH-PEG-DOX, and UCNPs@Au-DOX were measured to be −40.1, −4.1, +3.5, and +6.0 mV, respectively, indicating that the SH-PEG-DOX was successfully bonded to the cit-UCNPs@Au and resulted in UCNPs@Au-DOX (Fig. S4). The final UCNPs@Au-DOX nanocomposites are electropositive, making them more situable for cellular uptake.

image file: c7nr02280h-f1.tif
Fig. 1 TEM images of (a) NaYF4:Yb,Er@NaGdF4 (UCNPs), (b) cit-UCNPs, and (c) cit-UCNPs@Au; (d) HRTEM image of cit-UCNPs@Au, white circles: gold nanoparticles; (e) STEM-HAADF image and (f) selected area electron diffraction (SAED) pattern of UCNPs@Au-DOX.

Compared with other nanocomposite materials based on a combination of UCNPs and gold nanocrystals,43 in our case, the gold nanocrystals were grown in situ on the surface of the cit-UCNPs. This method of combination is very facile, and most importantly the gold nanocrystals closely combined with the UCNPs, implying that the two originally independent parts will not separate, which is beneficial for synergistic effects in actual biological applications. As shown in Fig. S5, the XRD pattern of the UCNPs@Au-DOX nanocomposites displays all of the crystal diffraction peaks of gold nanoparticles. The Bragg diffraction peaks at 2θ values of 38.24°, 44.94°, 64.74°, 77.58°, and 81.94° correspond to the (111), (200), (220), (311), and (222) planes of the gold nanocrystals.44,45 The other diffraction peaks are well indexed to the characteristic peaks of standard hexagonal phase β-NaYF4 (JCPDS no. 16-0334). These results further demonstrate that this facile method is effective to obtain nanocomposites of UCNPs@Au-DOX.

In addition, the attachment of SH-PEG-DOX to the cit-UCNPs@Au was further investigated by Fourier-transform infrared (FT-IR) spectroscopy. The FT-IR spectra of the UCNPs, citric acid, and cit-UCNPs are displayed in Fig. S6a. In the curve of the UCNPs, the peaks at 2926 and 2857 cm−1 are assigned to the symmetric and asymmetric stretching vibrations of methylene (–CH2) in the long alkyl chain of OA, and the band at 1567 cm−1 is attributed to the C[double bond, length as m-dash]O group, indicating that OA was coated on the surface of the UCNPs.46 Compared with the curve of citric acid, the new peak at 1645 nm for the cit-UCNPs suggests that cit-UCNPs were obtained. As shown in Fig. S6b, in comparison with the spectra of DOX and SH-PEG-HZ, the new peaks at 1578 and 1715 cm−1 in the curve of SH-PEG-DOX are attributed to –C[double bond, length as m-dash]N– and –CONH– in the hydrazone bond, respectively, which suggests that the SH-PEG-DOX prodrug was successfully synthesized.47 The new peak that appeared at 1709 cm−1 in the curve of the UCNPs@Au-DOX nanocomposites suggests the successful conjugation of SH-PEG-DOX and cit-UCNPs@Au, attributed to the strong Au–S bond.48 In addition, the visible absorption spectra of the cit-UCNPs, DOX, cit-UCNPs@Au, and UCNPs@Au-DOX are shown in Fig. S7a. Compared to the cit-UCNPs, the cit-UCNPs@Au nanoparticles display a wide absorption band from 400 nm to 700 nm. After linking with SH-PEG-DOX, the appearance of the characteristic peak of DOX in the UCNPs@Au-DOX nanocomposites indicates that UCNPs@Au-DOX was obtained. In addition, from the TGA results it can be calculated that the weight of the SH-PEG-DOX should be around 4.7% (Fig. S8).

The upconversion luminescence (UCL) spectra of the cit-UCNPs, cit-UCNPs@Au, and UCNPs@Au-DOX nanocomposites are shown in Fig. 2a. Under excitation at CW 980 nm, all of the nanomaterials show three characteristic bands at 521 nm, 540 nm, and 654 nm, attributed to the 2H11/24I15/2, 4S3/24I15/2 and 4F9/24I15/2 transitions of the Er3+ ion, respectively. However, upon crystal growth of gold nanocrystals on the surface of the cit-UCNPs, the UCL intensity of cit-UCNPs@Au decreased both in the green and red areas, as shown in Fig. 2a. This is attributed to the overlap between the UCL spectrum of the cit-UCNPs and the absorption spectrum of cit-UCNPs@Au, resulting in a luminescence resonance energy transfer (LRET) process (Fig. S7b). Photographs of the cit-UCNPs, cit-UCNPs@Au and UCNPs@Au-DOX nanocomposites are displayed in Fig. 2b. Furthermore, Fig. S9a and S9b show the UCL decay profiles of the 4S3/2 and 4F9/2 levels of Er3+ under excitation at 980 nm, respectively. The UCL lifetimes of the two levels of Er3+ ions of cit-UCNPs@Au are shorter than those of the cit-UCNPs, indicating that there is an energy transfer process between the cit-UCNPs and the gold nanocrystals.49 It is noteworthy that the decrease in UCL in the green area is stronger than that in the red area, because there is a wider overlap between the green UCL spectrum of the cit-UCNPs and the absorption spectrum of the cit-UCNPs@Au nanoparticles. After linking with SH-PEG-DOX, the UCL intensity of the UCNPs@Au-DOX nanocomposites showed a slight decrease.

image file: c7nr02280h-f2.tif
Fig. 2 (a) The upconversion luminescence (UCL) spectra of cit-UCNPs, cit-UCNPs@Au, and UCNPs@Au-DOX in water (400 μg mL−1); the insets are photographs of the cit-UCNPs, cit-UCNPs@Au, and UCNPs@Au-DOX (from left to right) under excitation with a 980 nm laser, at 1.5 W cm−2. (b) The corresponding photographs of (1) cit-UCNPs, (2) cit-UCNPs@Au, and (3) UCNPs@Au-DOX.

3.2. In vitro DOX release profiles of UCNPs@Au-DOX

Before the in vitro DOX release test, the drug loading content of UCNPs@Au-DOX was determined to be 5.6 wt% by using the calibration of the absorbance at 480 nm (Fig. 3a). The hydrazone bond is unstable in acidic conditions with a characteristic pH-responsive nature, causing the pH-dependent release of DOX from the UCNPs@Au-DOX nanocomposites due to the hydrolysis of this bond.50 Then, the drug release profiles of the UCNPs@Au-DOX nanocomposites were acquired in a simulated physiological PBS buffer environment at different pH values (pH = 4.5, 6.0, and 7.4), and the absorbance of DOX at 480 nm was used to calculated the released amount of DOX. As shown in Fig. 3b, at a physiological pH value (pH = 7.4) the UCNPs@Au-DOX nanocomposites were relatively stable and the cumulative release amount of DOX was 9.4% after 72 h. At pH = 6.0, the cumulative release amount of DOX was higher than that at pH = 7.4 and reached 18.9% after 72 h. At pH = 4.5, which is similar to the intracellular pH of cancer cells, the rapid and abundant release of DOX was observed due to the rapid hydrolysis rate of the hydrazone bond. After 48 h, the release of DOX reached a plateau and the cumulative release amount of DOX was as high as 47.0%. This is probably attributed to the reversible nature of the hydrazone bond, and the subsequent establishment of an equilibrium between free DOX and the UCNPs@Au-DOX nanocomposites. These results illustrate the feasibility of the UCNPs@Au-DOX nanocomposites for the release of intracellular DOX in chemotherapy.
image file: c7nr02280h-f3.tif
Fig. 3 (a) The absorption intensity (at 480 nm) as a function of DOX concentration (0–0.8 mg mL−1). (b) Release behavior of DOX from UCNPs@Au-DOX in PBS buffer solution (pH = 4.5, 6.0, and 7.4).

3.3. Photothermal conversion performance

The UCNPs@Au-DOX nanocomposites display a broad Vis-NIR absorbance in the 400–850 nm region, as shown in Fig. S10, suggesting that they are a potential candidate for application in PTT.51 First, the photothermal performance of UCNPs@Au-DOX was investigated in 5 mL sample bottles. Under continuous irradiation with an 808 nm laser (1.5 W cm−2) for 10 min, the temperature variation of UCNPs@Au-DOX with different concentrations was recorded as a function of time, as shown in Fig. 4a. The temperature of ultra-pure water, which served as a control, increased by less than 10.2 °C from room temperature of 22.3 °C during 10 min. With the increase of the concentration (50, 100, 200, and 400 μg mL−1) of the UCNPs@Au-DOX, the temperature of the solution rapidly increased in the first six minutes and then increased slowly until the temperature reached a plateau, and the improved temperatures (ΔT) in 10 min calculated from Fig. 4a were 13.0, 17.6, 26.3, and 33.5 °C, respectively, as shown in Fig. 4b. The results indicated that the UCNPs@Au-DOX can efficiently convert light energy (808 nm) to thermal energy. In addition, the photothermal conversion efficiency (η) was calculated to be 12% by using a previously reported method described in the ESI according to the obtained data (Fig. 4c and d). Furthermore, Fig. 5 shows the infrared thermal images of the different concentrations of UCNPs@Au-DOX nanocomposites at the time points of 0, 1, 2, 3, 4, 5, 6, 7, and 8 min with continuous irradiation with an 808 nm laser (1.5 W cm−2). As vividly shown by the intensity bar ranging from 11.8 °C to 95.7 °C, with the increase of concentration, the temperature of the UCNPs@Au-DOX nanocomposites increased rapidly. As we know, cancer cells can be effectively killed at temperatures higher than 42 °C, as they are more sensitive to heat in comparison with normal cells.52 Thus, the UCNPs@Au-DOX nanocomposites are a potential photothermal therapy candidate. In addition, after exposure to an 808 nm laser for 8 min, the temperatures of the UCNPs@Au-DOX nanocomposites nearly became stable, so 8 min was selected as the optimal laser irradiation time in the following cytotoxicity assessment.
image file: c7nr02280h-f4.tif
Fig. 4 (a) Temperature profiles of pure water and water solutions of the UCNPs@Au-DOX nanocomposites at different concentrations (50, 100, 200, and 400 μg mL−1, containing 106.66, 53.01, 26.17, and 13.03 μg mL−1 gold, respectively) as a function of irradiation time (0–10 min) under 808 nm laser irradiation (1.5 W cm−2). (b) Plot of temperature change (ΔT) over a period of 10 min versus different concentrations of UCNPs-Au-DOX. (c) The monitored temperature change over 20 min with UCNPs-Au-DOX (400 μg mL−1) after 808 nm laser irradiation (1.5 W cm−2) for 10 min, following the laser being turned off. (d) Time constant for heat transfer from the system calculated to be τs = 221.1 s by using the linear time data from the cooling period (after 600 s) versus the negative natural logarithm of the driving force temperature, which is obtained from the cooling profile in Fig. 3c.

image file: c7nr02280h-f5.tif
Fig. 5 Infrared thermal images at the time points of 0, 1, 2, 3, 4, 5, 6, 7, and 8 min of the different concentrations of UCNPs@Au-DOX nanocomposites under 808 nm laser irradiation (1.5 W cm−2).

3.4. In vitro cytotoxicity assessment

The effective DOX release in PBS buffer solution and strong energy transfer from an 808 nm laser of the UCNPs@Au-DOX nanocomposites encouraged us to develop a potential synergistic effect of chemotherapy and photothermal therapy for cancer cells with an 808 nm continuous wavelength (CW) laser. The cytotoxicity of the nanomaterials was investigated. Before the cell assessment, the dispersion stabilities of cit-UCNPs@Au and UCNPs@Au-DOX were investigated in PBS and DMEM culture solution. As shown in Fig. S11, there is no obvious aggregation during 24 h, which suggests that these materials are suitable for bioapplications. Furthermore, a series of experiments were conducted using HeLa cells incubated with cit-UCNPs@Au and UCNPs@Au-DOX nanocomposites with different concentrations ranging from 50 to 200 μg mL−1, in which corresponding the content of free DOX served as a control.

As shown in Fig. 6, the toxicity of cit-UCNPs@Au was first evaluated, and the results indicate that the viabilities of HeLa cells were nearly 100% after incubation with cit-UCNPs@Au, even at the highest concentration (200 μg mL−1), for 24 h. This shows that the cit-UCNPs@Au nanoparticles possess excellent biocompatibility. It is worth noting that upon irradiation with an 808 nm laser, the cit-UCNPs@Au nanoparticles with low concentration (50 μg mL−1) show no obvious toxicity to HeLa cells, which is in agreement with the temperature profiles in Fig. 4a, implying that the temperature is insufficient to damage the cells. After linking with DOX, the lower viability of UCNPs@Au-DOX is ascribed to the chemical toxicity of the drug released from the nanocomposites, indicating a synergistic effect of chemotherapy and photothermal therapy. As for high concentrations of cit-UCNPs@Au and UCNPs@Au-DOX (such as 100 and 200 μg mL−1), the PTT effects (with laser irradiation) play a dominant role in comparison with the chemotherapy (drug release). The cit-UCNPs@Au nanoparticles (200 μg mL−1) under 808 nm laser irradiation displayed strong phototoxicity to HeLa cells with a low cell viability of 18.6%. Furthermore, the cell viability decreases markedly to 3.0% for the UCNPs@Au-DOX nanocomposites (200 μg mL−1) with 808 nm laser irradiation, much lower than that of the UCNPs@Au-DOX nanocomposites without laser irradiation (65.0%). The data above indicate that the cit-UCNPs@Au nanoparticles exhibit a good PTT effect for cancer cells when the concentration is higher than 100 μg mL−1. Of importance is that the final UCNPs@Au-DOX nanocomposites (with a concentration of 200 μg mL−1) can afford excellent cytotoxicity to cancer cells and effective cancer treatment efficiency under irradiation with an 808 nm laser, which results from the synergistic effect of chemotherapy and photothermal therapy.

image file: c7nr02280h-f6.tif
Fig. 6 Cytotoxicity assays of HeLa cells incubated with various concentrations of free DOX, cit-UCNPs@Au, and UCNPs@Au-DOX without and with 808 nm laser irradiation for 24 min (1.5 W cm−2, 8 min break after each 8 min irradiation). HeLa cells in normal culture medium serve as the control group.

In addition, a series of live/dead cell assays were carried out to monitor the chemotherapy and photothermal effect by staining the HeLa cells with calcein AM and PI, which can emit green and red fluorescence in live and dead cells, respectively. As shown in the control group of Fig. 7, upon irradiation with an 808 nm laser, the HeLa cells still showed bright green emission, indicating that the 808 nm laser has no effect on the viability of HeLa cells. When treated only with cit-UCNPs@Au (200 μg mL−1), the HeLa cells emit bright green fluorescence in the entire well region, suggesting that the cit-UCNPs@Au nanoparticles are biocompatible. After irradiation with an 808 nm laser, a large number of cells were dead due to the fact that the effective transformation of laser energy to thermal energy leads to cell death. In contrast, incubation with 200 μg mL−1 UCNPs@Au-DOX leads to a small proportion of dead cells (with weak red emission). Furthermore, the total number of cells is reduced, which is attributed to the pH-responsive drug release of UCNPs@Au-DOX in the acidic environment of the lysosome. After irradiation with an 808 nm laser, nearly all of the cells died and exhibited bright red fluorescence in the entire well region, which effectively proves that the UCNPs@Au-DOX is an excellent candidate for cancer treatment due to the synergistic effect of chemotherapy and photothermal therapy.

image file: c7nr02280h-f7.tif
Fig. 7 Fluorescence microscopy images of calcein AM/PI-stained HeLa cells (green represents living cells, red represents dead cells) incubated with 200 μg mL−1 cit-UCNPs@Au and UCNPs@Au-DOX, respectively, without and with 808 nm laser irradiation (1.5 W cm−2, 24 min, 8 min break after each 8 min irradiation). HeLa cells in normal culture medium serve as the control group. After irradiation with an 808 nm laser, the HeLa cells still show bright green emission, indicating that the 808 nm laser has no effect on the viability of HeLa cells. Scale bar: 100 μm.

3.5. In vitro CLSM imaging of UCNPs@Au-DOX in HeLa cells

Based on the great stability of UCNPs@Au-DOX, cellular internalization localization was investigated by CLSM imaging to demonstrate that the nanocomposites could serve as a visual tracking agent of anti-cancer drugs under CW excitation at 980 nm. As shown in Fig. S12, HeLa cells, after being incubated with cit-UCNPs@Au (200 μg mL−1) for 0.5, 1, 2, and 4 h at 37 °C, respectively, all displayed merged UCL imagings of green (500–600 nm) and red (600–700 nm) channels collected as a series along the Z optical axis (Z-stack). It can be noted that the intensity of UCL became brighter and brighter over time, suggesting that more and more cit-UCNPs@Au nanoparticles were uptaken by HeLa cells. Similar imaging phenomena were observed in the HeLa cells incubated with the UCNPs@Au-DOX nanocomposites. As shown in Fig. 8, after the HeLa cells were incubated with UCNPs@Au-DOX, it can be observed that more and more UCNPs@Au-DOX nanocomposites were uptaken by the HeLa cells from 2 to 4 h, deduced from the green and red UCL signals (as the arrows show). It should also be noted that the enhancement of the UCL signals might also come from the intracellular DOX release from the UCNPs@Au-DOX nanocomposites. Because the release of DOX weakens the LRET effect between the UCNPs and DOX, the UCL intensities gradually increase upon drug release.53
image file: c7nr02280h-f8.tif
Fig. 8 Confocal laser scanning microscopy (CLSM) images of HeLa cells after incubation with UCNPs@AuaDOX for 2 and 4 h; the rose red emission from DOX was collected at 550–700 nm under excitation of 488 nm; upconversion luminescence (UCL) emission was collected by a green UCL channel at 500–600 nm and a red channel at 600–700 nm, λex = 980 nm, 500 mW.

In addition, the rose red emission from DOX increased strongly with an increase in incubation time, suggesting that the DOX was released from the UCNPs@Au-DOX nanocomposites gradually in the HeLa cells. In addition, the different permeability of nuclear membrane to DOX and the UCNPs plays a significant role in cell uptake. Generally, it is much easier for DOX to enter the nucleus of the cells through the nuclear pores on the nuclear membrane than the nanoparticles.54 Thus, with prolonged incubation time, the DOX that was released from the UCNPs@Au-DOX nanocomposites tended to enter the cell nucleus, which resulted in some of the DOX signal (rose red) not overlapping completely with the UCL signal (yellow). Therefore, the results mentioned above indicate that the UCNPs@Au-DOX nanocomposites are desirable candidates for bioimaging.

3.6. Magnetic resonance imaging of UCNPs@Au-DOX

Noteworthily, bioimaging-guided therapy is playing an increasingly important role in the cancer therapy field. Magnetic resonance imaging (MRI) is a valuable and noninvasive medical imaging technique with high organ resolution, and has been widely applied for biological tissues.55 In order to use the UCNPs@Au-DOX nanocomposites for bioimaging-induced therapy, the core of the NaYF4:Yb,Er nanoparticles was coated with an NaGdF4 shell, which can not only improve the UCL intensity of the final nanocomposites but also provide them with suitable properties for T1-weighted MRI. The presence of the highest number of unpaired f electrons with parallel spin for the Gd3+ ion leads to a high magnetic moment of 7.94μB and slows electron relaxation, which results in a line broadening effect on the neighboring water protons.56 In this case, the T1 values were recorded on a 3.0 T MRI scanner, and the concentrations of Gd3+ were determined by ICP-AES. As shown in Fig. 9a, with increasing Gd3+ concentration, the brightness of the T1-weighted MRI increased, which can be vividly observed in the color-mapped images. Fig. 9b shows that the value of 1/T1 increased as a function of Gd concentration. A linear curve was obtained with a slope of 3.0595 (3.06) mM−1 S−1, confirmed to be the longitudinal relaxivity value (r1), which is high in comparison with other NaGdF4-based nanomaterials encapsulated with polymers or other shells.57 Therefore, it can be deduced that the UCNPs@Au-DOX nanocomposites could be introduced as potential T1-weighted MRI contrast agents.
image file: c7nr02280h-f9.tif
Fig. 9 (a) T1-Weighted and color-mapped magnetic resonance (MR) images for various Gd3+ concentrations of UCNPs@Au-DOX nanocomposites. (b) Relaxation rate r1 (1/T1) plotted against the different Gd3+ concentrations of UCNPs@Au-DOX.

4. Conclusions

In summary, we report a novel and facile method to synthesize multifunctional nanocomposites possessing imaging-guided synergistic effects of chemotherapy and photothermal therapy. For the first time, gold nanocrystals were grown in situ on the surface of citric acid-modified UCNPs (cit-UCNPs) with a spherical morphology, where the cit-UCNPs served as a stabilizer and reducing agent in the in situ growth process. Benefiting from the effective photothermal effect of cit-UCNPs@Au, the SH-PEG-DOX prodrug, which acts as a chemotherapeutic agent, was linked with cit-UCNPs@Au to obtain the final UCNPs@Au-DOX nanocomposites. A hydrazone bond was introduced into the structure of the prodrug, which is beneficial for pH-responsive drug release in the acidic microenvironment of cancer cells. The in vitro cell viability assessment indicated that the UCNPs@Au-DOX nanocomposites exhibit the synergistic effect of chemotherapy and photothermal therapy to enhance the treatment efficiency with very low cell viability. In addition, confocal laser scanning microscope (CLSM) images of UCNPs@Au-DOX in HeLa cells and T1-weighted MR imaging suggest that the UCNPs@Au-DOX nanocomposites may open up potential routes for bioimaging-guided cancer therapy in biological and biomedical applications.


We are grateful for the financial support from the Key Program for International Science and Technology Cooperation Projects of Ministry of Science and Technology of China (No. 2016YFE0114800), the National Natural Science Foundation of China (Grant No. 21571125 and 21231004), and the Science and Technology Commission of Shanghai Municipality (14520722200). We are also grateful to the Instrumental Analysis & Research Center of Shanghai University.

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Electronic supplementary information (ESI) available: The formulas of the photothermal conversion effect of UCNPs@Au-DOX. HRTEM image of cit-UCNPs@Au. The energy dispersive X-ray (EDX) spectrum and element mapping of cit-UCNPs@Au. DLS of the cit-UCNPs, cit-UCNPs@Au, and UCNPs@Au-DOX in water. The zeta potential of the UCNPs in cyclohexane, and the cit-UCNPs, cit-UCNPs@Au, SH-PEG-DOX and UCNPs@Au-DOX in water. XRD patterns of the cit-UCNPs and UCNPs@Au-DOX. FT-IR spectra of the UCNPs, citric acid, cit-UCNPs, DOX, SH-PEG-HZ, SH-PEG-DOX and UCNPs@Au-DOX. Visible absorption spectra of the UCNPs, cit-UCNPs, DOX, cit-UCNPs@Au and UCNPs@Au-DOX and spectral overlap between the UCL spectrum of UCNPs@Au-DOX and the absorption spectrum of DOX. The UCL decay profile of the 4S3/2 and 4F9/2 level of Er3+ under the excitation of a 980 nm laser. TGA traces of cit-UCNPs@Au and UCNPs@Au-DOX. Bright-field photographs of cit-UCNPs@Au and UCNPs@Au-DOX in PBS and DMEM culture solution. Three-dimensional confocal luminescence reconstructions of HeLa cells after incubation with UCNPs@Au-DOX for 0.5, 1, 2, and 4 h. See DOI: 10.1039/c7nr02280h

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