Lanthanide-doped upconversion nanoparticles complexed with nano-oxide graphene used for upconversion fluorescence imaging and photothermal therapy

Po Li a, Yue Yan b, Binlong Chen c, Pan Zhang a, Siling Wang b, Jing Zhou a, Haiming Fan *d, Yiguang Wang *c and Xiaonan Huang *a
aDepartment of Chemistry, Capital Normal University, 105 West 3rd Ring North Rd, Beijing 100048, PR China
bDepartment of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning Province 110016, PR China
cBeijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing, China
dSchool of Petroleum Engineering, China University of Petroleum (East China), Qingdao 266580, Shandong Province, PR China

Received 30th November 2017 , Accepted 26th January 2018

First published on 5th February 2018

In recent years, multifunctional nanoparticles have attracted much research interest in various biomedical applications such as biosensors, diagnosis, and drug delivery systems. In this study, we report an NIR imaging diagnosis and therapy nanoplatform which is developed by complexing upconversion nanoparticles (UCNP@OA) NaLuF4:Er3+,Yb3+ with nanographene oxide (NGO). The obtained nanocomposites UCNP@NGO showed excellent stability and low cell toxicity, which not only acted as upconversion luminescence (UCL) probes for tumor imaging, but also served as therapy agents by converting laser energy into thermal energy for photothermal therapy (PTT) with high photothermal conversion efficiency. This work highlights the potential of UCNP@NGO nanocomposites as an integrated theranostic nanoplatform for the UCL image combinatorial PTT of cancer, providing a promising candidate for clinical antitumor treatments.

1. Introduction

With population growth and aging, cancer has become a major cause of death to human beings.1 Among all cancer treatments, photothermal therapy (PTT) has attracted widespread interest with the advantages of harmless treatment, minimal invasion, high efficiency and deep tissue penetration.2–5 The principle of this novel approach is to generate heat from near-infrared light by employing photo-absorbing agents, which will result in the overheating of cancer cells.6,7 Nano graphene oxide (NGO), with monolayers or a few layers of sp2-bonded carbon atoms, has been widely applied as one of the most effective nanomaterials in photothermal therapy applications due to its excellent thermal properties. NGO can absorb NIR radiation and transfer it into thermal energy because of its delocalized electron arrangement,8,9 resulting in a temperature increase of the tumor tissue and structural changes in the cell.10 So far, a lot of work about the photothermal therapy of cancer cells with NGO has been reported and the excellent water solubility and biocompatibility have been illustrated.11–18

Precisely the irradiation of PTT agents can efficiently improve the cancer therapy effect. To provide precise guidance of cancer treatments and track the photothermal agents in vivo, imaging-guided PTT is becoming a promising method,19–22 and lanthanide-doped rare-earth upconversion nanoparticles (UCNPs) have attracted enormous attention as a new generation of luminescent nanoprobes in the past few decades.23–26 Compared to conventional materials, UCNPs have shown several significant advantages in biomedical imaging, such as a sharp emission bandwidth, long lifetime, tunable emission, high photostability, and low cytotoxicity.27–31 More importantly, the near-infrared (NIR) excitation of UCNPs within the “optical transmission window” of biological tissues significantly enhanced the penetration depths (700–1000 nm) and minimized the background autofluorescence, photobleaching as well as photodamage to biological specimens.32,33 Benefiting from such unique properties, UCNPs have successfully been applied as the probes to identify the location of cancer cells, monitor the biodistribution of nanocomposites and assess the therapeutic efficacy.34–36

Herein we developed a multifunctional UCNP@NGO complexed nanocomposite for upconversion fluorescence imaging and photothermal therapy, which combined the PTT agent NGO and upconversion luminescence probe UCNPs via a carboxyl group. The resulting theranostic nanoplatform exhibited both a high upconversion luminescence intensity for imaging and an improved therapeutic effect owing to PTT. This novel nanocomposite integrated UCL imaging and PTT into a single nanoplatform, which could act as a theranostic probe for the UCL image combinatorial PTT of cancer (Scheme 1). Since both imaging and PTT treatments in this nanoplatform are stimulated by light, only the tumor lesion would be exposed, and compared to conventional cancer therapies, this system exhibited remarkable advantages in terms of enhancing the cancer killing specificity as well as reducing side effects. Moreover, a synergistic effect of the combined UCL imaging diagnosis and photothermal therapy was confirmed and expected to decrease the dosage-limiting toxicity and tissue damage by over-heating and to improve the therapeutic efficiency.

image file: c7bm01113j-s1.tif
Scheme 1 Illustration of the complexation of UCNP@NGO as a theranostic agent for near-IR imaging and photothermal therapy.

2. Experimental

2.1 Material preparation

NH4F, NaOH, ethanol, and cyclohexane were purchased from Sinopharm Chemical Reagent Co., China. Oleic acid (OA) and octadecene (ODE) were purchased from Alfa Aesar Ltd. LnCl3 (>99.9%, Ln = Lu, Yb, and Er) was purchased from Aladdin Reagent Co., China. Graphene oxide powder (GO) (>99%) was purchased from JCNANO (Nanjing, China). All other chemical reagents were of analytical grade and directly used without further purification. Deionized water was used throughout the experiments.

2.2 Synthesis of UCNP@OA

Monodisperse OA-capped upconversion luminescence NaLuF4:Yb3+,Er3+ nanocrystals (UCNP@OA) were synthesized through a facile co-precipitation protocol as reported.37,38 A mixture of 0.8 mmol of LuCl3, 0.18 mmol of YbCl3, 0.02 mmol of ErCl3, 6 mL of oleic acid and 15 mL of ODE was added into a 100 mL flask. At 40 °C, the flask was evacuated and then filled with nitrogen gas to remove oxygen, and this process was repeated three times. The temperature of the resulting mixture was continuously increased to 150 °C until the liquid boiled. After the solution was cooled down to room temperature, 2.5 mmol of NaOH and 4 mmol of NH4F were added dropwise into the flask. Under an argon atmosphere, the solution was heated to 70 °C until the liquid was homogeneous, and then continued to be heated to 300 °C for 1 h. The reaction mixture was then cooled down to room temperature. The UCNC@OA was precipitated by the addition of ethanol, and collected by centrifugation, then washed with a (1[thin space (1/6-em)]:[thin space (1/6-em)]1 v/v) hexane/ethanol solution three times and redispersed in 10 mL of cyclohexane.

2.3 Preparation of nanosized graphene oxide (NGO)

GO powder (15.0 mg) was dissolved in ammonium hydroxide (30 mL), and then exfoliated by using an ultrasonic cell disruptor (95% amplitude, 800 W, 4 h) until the entire size distribution was below 120 nm. Centrifugation (12[thin space (1/6-em)]000 rpm, 20 min) was performed to remove the un-exfoliated large GO sheets. The prepared NGO showed a narrow size distribution of around 120 nm which is characterized by dynamic light scattering.

2.4 Synthesis of UCNP@NGO

The UCNP@NGO nanocomposites were prepared by a modified-ligand exchange strategy. Purified UCNP@OA solution (50 mg) was added dropwise into the NGO solution (30 mL, 0.5 mg mL−1) by using an ultrasonic cell disruptor (95% amplitude, 800 W, 4 h). After stirring for another 12 h, the solution was centrifuged (12[thin space (1/6-em)]000 rpm, 20 min) twice to remove excess free NGO nanosheets. Then the precipitates were collected and redispersed with 15 mL of water.

2.5 Characterization

The morphological features of the UCNP@OA and UCNP@NGO were characterized with a Hitachi 7650 transmission electron microscope (TEM). Fourier Transform infrared spectroscopy (FTIR) analysis was carried out with a PerkinElmer FT-IR spectrometer. The hydrodynamic size was determined with a Malvern Zetasizer ZS90 (Malvern Instruments Ltd, UK). Thermogravimetric analysis (TGA) of UCNP@OA, NGO and UCNP@NGO was carried out with a Q50 thermogravimetric analyzer under a nitrogen flow with a heating rate of 20 °C min−1. The upconversion fluorescence spectra were recorded using a Maya 2000 Pro spectrophotometer (Shanghai Oceanhood Opto-electronics Tech Co. Ltd) equipped with an external 980 nm laser instead of an internal excitation source.

2.6 In vitro cytotoxicity studies

The cytotoxicity of UCNP@NGO was determined by the MTT assay with mouse breast cancer cells (4T1 cells, purchased from the Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College). The 4T1 mammary carcinoma cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. The 4T1 cells were seeded into a 96-well tissue culture plate at a seeding density of 5000 cells per well in a CO2 (5%) incubator at 37 °C. After incubation for 24 h, the pristine DMEM medium was replaced with 200 μL of NGO (0, 50, 100, 200, and 400 μg mL−1) or UCNP@NGO (0, 50, 100, 200, and 400 μg mL−1), respectively. For the control experiment, the medium was replaced with a fresh culture medium. After incubation for another 24 h, the viability of the cells was measured by using the MTT assay.

2.7 In vitro PTT treatments

The 4T1 cells were seeded in 96-well plates (5000 cells per well) and incubated at 37 °C under a humidified 5% CO2 atmosphere for 24 h. The cells were then incubated with NGO (0, 50, 100, 200, and 400 μg mL−1) and UCNP@NGO (0, 50, 100, 200, and 400 μg mL−1) in a medium supplemented with 10% FBS for 24 h at 37 °C. After being rinsed with PBS, the cells in the irradiation groups were exposed to an 808 nm laser with an energy density of 2 W cm−2 for 5 min. The cells in the dark groups were rinsed with blank culture medium without laser irradiation. All the cells were rinsed with PBS, and then 200 μL of MTT (500 μg mL−1) solution was added to each well and the plate was incubated for an additional 4 h. After the addition of dimethyl sulfoxide (DMSO, 200 μL per well), the assay plate was allowed to stand at room temperature for 30 min and the standard MTT assay was performed to evaluate cell viability, which was measured by means of background subtraction at 540 nm on a Thermo Scientific multiskan FC microplate reader.

To investigate the effect of the irradiation time, all the cells were incubated with NGO (200 μg mL−1) or NGO@UCNP (200 μg mL−1) in a medium supplemented with 10% FBS for 24 h at 37 °C. After being rinsed with PBS, the cells were exposed to an 808 nm laser with an energy density of 2 W cm−2 for different irradiation times (0, 1, 3, 5, and 10 min). The following experimental steps are the same as mentioned above.

The PTT effects of NGO and UCNP@NGO were further verified on 4T1 cells using calcein AM and PI co-staining. 24 h after laser irradiation, the cells were incubated with calcein-AM (2 μM) and PI (1 μM) in 1 mL of PBS for 30 min and observed by inverted fluorescence microscopy.

2.8 In vivo PTT therapy

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Peking University and approved by the Animal Ethics Committee of Peking University. 4T1 breast tumor-bearing BALB/c mice were administered separately with PBS, NGO and UCNP@NGO. A vernier caliper was utilized to measure the tumor size, and the tumor volume was calculated using the following formula: V (mm3) = [length × (width)2]/2. When the tumors reached a size of 100 mm3, the mice were randomly assigned into six groups and this time point was designated as day 0. The mice (n = 5) were treated with 100 μL of PBS, NGO and UCNP@NGO, respectively. The PBS, NGO and UCNP@NGO were injected around the tumors and surrounded the tumors for the maximum benefit of treatment. Once injected, the mice were anaesthetized and the tumor areas were irradiated with an 808 nm NIR laser (1 W cm−2) for 5 min. To monitor the photothermal effects in vivo, the infrared thermal imaging and temperature changes of the tumors were simultaneously recorded with an infrared thermal imaging camera.

3. Results and discussion

3.1 Preparation and characterization of UCNP@NGO

NaLuF4:Yb3+,Er3+ nanoparticles were synthesized following a previously reported protocol with some modifications.37,38 Due to the OA-capped surface, the prepared nanoparticles are hydrophobic and can be steadily dispersed in nonpolar organic solvents, such as cyclohexane and chloroform. The transmission electron microscopy (TEM) images of the NaLuF4:Yb3+,Er3+ nanoparticles are shown in Fig. 1(a), which demonstrate the monodisperse nanoparticles showing a uniform size of approximately 55 nm. X-ray diffraction (XRD) analysis is used to determine the crystallinity of the UCNP@OA. These main diffraction peaks of the UCNP@OA sample agree with the standard hexagonal phase structure of bulk β-NaLuF4 (JCPDS 27-07-27) very well (Fig. S1).
image file: c7bm01113j-f1.tif
Fig. 1 TEM image of UNCP@OA (a) and UCNP@NGO (c); (b) histograms of the diameter of UNCP@OA; (d) FTIR-spectra and (e) thermogravimetric analysis (TGA) of the white solid residue UCNP@OA (red line), free NGO (blue line), and UCNP@NGO (black line).

The NGO was prepared by using an ultrasonic cell disruptor and coordinated to the surface of the UCNP in a basic environment in aqueous solution through the carboxylic acid group on the NGO by the ligand exchange strategy. The efficient coordination of UCNPs and NGO were demonstrated by the IR absorption of nanoparticles before and after complexation as shown in Fig. 1(d). For UCNP@OA, the multipeak bands at 2925 and 2855 cm−1 were assigned to the stretching vibrations of –CH2–. After the complexation, the peaks at 2925 and 2855 cm−1 disappeared in UCNP@NGO, which revealed that OA groups on the surface of the UCNPs were completely replaced by NGO. The exact amount of NGO in each nanoparticle was determined by thermogravimetric analysis (TGA, Fig. 1(e)). The TGA curves of the UCNP@OA and UCNP@NGO are illustrated in Fig. 1(e), where the weight loss of OA and NGO on the surface of UCNPs was calculated to be 5% and 34%, respectively. The significant difference in the weight loss further confirmed that NGO successfully replaced OA on the surface of UCNPs by the ligand exchange process, which is in good agreement with the FTIR results.

3.2 Photothermal performance of the products

In order to investigate the photothermal performance, the UCNP@NGO with 0.5 mg mL−1 concentration was irradiated with an 808 nm NIR laser (2.0 W cm−2) for 5 min and the system temperature was monitored. As shown in Fig. 2(a), upon laser irradiation, the control sample pure DI water showed negligible changes, however, a pronounced increase in temperature could be observed for both free NGO and UCNP@NGO suspensions. Specifically, the temperature of the UCNP@NGO suspension at 0.5 mg mL−1 could be elevated by ∼63 °C after 5 min irradiation, while free NGO increased by only ∼55 °C, which demonstrates that UCNP@NGO shows higher photothermal conversion efficiency compared with free NGO. As we all know, a temperature over 43 °C can destroy cancer cells due to the lower heat tolerance, therefore both the free UCNP and UCNP@NGO can act as promising candidates in PTT. Next, the photothermal conversion efficiency (η) of the free UCNP and UCNP@NGO was measured using the method reported in the literature.39 The NGO and UCNP@NGO aqueous solutions were irradiated with the 808 nm laser for 5 min. Then the laser was turned off, and the dispersions were naturally cooled to ambient temperature. Based on the obtained data in Fig. 2(c) and (d), the conversion efficiency of NGO and UCNP@NGO were calculated through the following formula:
image file: c7bm01113j-t1.tif
where h is the heat transfer coefficient; S is the surface area of the quartz cuvette; Tmax and Tsurr refer to the maximum system temperature and initial temperature, respectively; Qs is the baseline energy input of the quartz cuvette and DI water without the nanoparticles; I is the 808 nm laser power; Aλ is the absorbance of the dispersion at 808 nm. According to the equations, the photothermal conversion efficiency of UCNP@NGO was determined to be ∼40%, which is comparable to the efficiency of NGO ∼35%.

image file: c7bm01113j-f2.tif
Fig. 2 (a) Temperature elevation and (b) IR thermal images of water, NGO and UCNP@NGO at various irradiation times upon NIR irradiation; the temperature change of (c) NGO and (d) UCNP@NGO over four laser ON/OFF cycles.

The strong photothermal conversion properties of the UCNP@NGO also provided high contrast for infrared thermal (IRT) imaging as shown in Fig. 2(b); as the irradiation time increased, the color of the photothermal images continuously changed from blue (corresponding to low temperature) to bright yellow (corresponding to high temperature). These results indicated that the UCNP@NGO samples could absorb and convert NIR light to a substantial amount of heat energy. With the imaging intensity relying on the irradiation time, the IRT imaging modal can be used to identify the UCNP@NGO location and provide real-time monitoring on the PTT process.

However, the photothermal conversion of the free NGO and UCNP@NGO was demonstrated by four repeated irradiation cycles (Fig. 2(c) and (d)). The free UCNP and UCNP@NGO solutions were irradiated with NIR irradiation for 300 s, and then naturally cooled at room temperature. This cycle was repeated four times. The temperature was continuously recorded with a NIR camera. For UCNP@NGO, the photothermal effect shows no attenuation (Fig. 2(d)). However, for free NGO, attenuation with 2 °C could be observed for every cycle, and the temperature could only reach 50 °C for the fourth cycle which is about 5 °C lower than 55 °C in the first cycle. The excellent photostability of UCNP@NGO was much better than that of free NGO. These results indicated that the UCNP@NGO with high photothermal conversion efficiency and photostability could be used as a photothermal agent.

3.3 In vitro and in vivo UCL imaging of the UCNP@NGO

The upconversion luminescence properties of UCNP@OA and UCNP@NGO were studied under the excitation at 980 nm. As shown in the photograph inset of Fig. 3(a), green light could be observed when excited with a 980 nm laser. As illustrated in the UCL spectra, both UCNP@OA and UCNP@NGO in water exhibited three characteristic intense emission bands at 520, 540, and 653 nm derived from the 2H11/24I15/2, 4S3/24I15/2, and 4F9/24I15/2 transitions of Er3+, respectively, where the dopant Yb3+ acted as an NIR absorber and efficiently transferred energy to the emitter Er3+. The UCL emission intensity of UCNP@NGO was a little less than that of UCNP@OA which was most likely induced by the fluorescence quenching of NGO and UNCP. The photostability of the UCNP@NGO was also examined with time when excited with a 980 nm laser. As shown in Fig. 3(b), no obvious intensity decline of UCL emission of UCNP@NGO could be observed within 30 min, which indicated that UCNP@NGO exhibited no photobleaching with continuous excitation.
image file: c7bm01113j-f3.tif
Fig. 3 (a) UCL spectrum and digital pictures of UCNP@OA and UCNP@NGO dispersed in water solution under the excitation at 980 nm (the same concentration 1 mg mL−1); (b) photostability of UCNP@NGO dispersed in water solution under the excitation at 980 nm; (c) in vivo upconversion luminescence image under the excitation of a 980 nm laser.

As an imaging agent, the performance of UCNP@NGO was investigated by in vivo UCL imaging with intratumoral injection of UCNP@NGO in a white mouse under the excitation at 980 nm. As shown in the in vivo UCL image of the white mouse, a strong UCL signal was easily detected on the tumor of the mouse's back (Fig. 3(c)). The SNR of the image was calculated by measuring the mean intensities of UCL. The mean intensity of UCL is about 72. The SNR of the in vivo UCL imaging is calculated to be about 6.

3.4 In vitro cytotoxicity

Prior to the theranostic application of the UCNP@NGO nanoplatform, the in vitro cytotoxicity of free NGO and UCNP@NGO was assessed using a traditional MTT assay. Fig. 4(a) shows the in vitro cell viability of mouse breast cancer cells (4T1) incubated with UCNP@OA, free NGO and UCNP@NGO at different concentrations ranging from 0 to 400 μg mL−1 for 24 h. The cell viabilities for all the samples were higher than 90%, even at the high concentration of 400 mg mL−1. These data show satisfactory results for the in vitro non-cytotoxicity of free NGO and UCNP@NGO. The excellent biocompatibility and low cytotoxicity imply that UCNP@NGO has the potential to serve as a theranostic probe for simultaneous UCL imaging and PTT of cancer.
image file: c7bm01113j-f4.tif
Fig. 4 In vitro PTT of UCNP@NGO. (a) In vitro cytotoxicity of 4T1 cells; (b) cell viability of 4T1 cells with an 808 nm (2 W cm−2) laser at different concentrations under 5 min irradiation and (c) at different irradiation times at a concentration of 200 μg mL−1, treated with UCNP@OA, NGO or UCNP@NGO for 24 h; (d) inverted fluorescence microscopy images of A549 cells stained with PI and calcein-AM after incubation with 200 μg mL−1 of nanoparticles and NIR laser irradiation at different times. The red channel images were obtained from PI (ex/em, 535/617 nm) while the green channel images were obtained from calcein-AM (ex/em, 495/515 nm), and (e) flow cytometric analysis.

Cell destruction by PTT was further studied with 4T1 cells with free NGO and UCNP@NGO. The cells were incubated with free NGO and UCNP@NGO at a series of concentrations, and then irradiated with an 808 nm laser. The growth inhibition of the cells was strongly dependent on the concentration of free NGO and UCNP@NGO. Both the concentration-dependent and irradiation-dependent manners were investigated. The cell with PBS buffer or UCNP@OA was used as the control experiment and exhibited negligible toxicity to 4T1 cells under laser irradiation (Fig. 4(b) and (c)). As expected, Fig. 4(b) and (c) show that the viability of the 4T1 cells treated with free NGO or UCNP@NGO decreases with the increasing concentration and irradiation time. For instance, under 808 nm laser illumination, the cell viability in two UCNP@NGO groups is 9.1 ± 2.2% (400 μg mL−1 for 5 min) and 2.3 ± 1.9% (200 μg mL−1 for 10 min), respectively.

The photothermal ablation ability of free NGO and UCNP@NGO on the 4T1 cells was further confirmed by using inverted fluorescence microscopy imaging with calcein-AM and PI staining. Calcein-AM is a cell-permeable dye that labels living cells with green fluorescence, whereas PI is a cell impermeable dye that only labels dead cells with red fluorescence. As shown in Fig. 4(d), cells in free NGO and UCNP@NGO laser groups exhibited intense red fluorescence, indicative of dead cells, while the cells in the control group showed bright green fluorescence, indicative of living cells. At the initial time, sporadic cells died with the free NGO and UCNP@NGO concentration of 200 mg mL−1. After the irradiation, as a contrast, a negligible decrease in the cell viability was observed for the control group under 808 nm laser radiation after 10 min. However, the free NGO and UCNP@NGO treated cells showed time-dependent reduced viability at a power density of 2 W cm−2 after NIR exposure. In accordance with the inverted fluorescence microscopy results, the fluorescence results directly revealed that almost no cells survived while being exposed to NIR irradiation with the incubation of UCNP@NGO at a power density of 2 W cm−2.

In addition, quantitative analysis of cell death in each treatment group was achieved by using flow cytometry (Fig. 4(e)). The apoptosis rate of the cells reached as high as 73.6% in free NGO and 99.2% in the UCNP@NGO laser group. The UCNP@NGO group indicated near-total cell damage via a photothermal effect, which is much higher than the free NGO. Altogether, the in vitro cell studies consistently demonstrated that UCNP@NGO were capable of efficiently ablating cancerous cells via the photothermal effect and were little toxic without laser irradiation. Also, the in vitro cell studies demonstrated that UCNP@NGO nanoparticles possessed excellent biocompatibility and were capable of efficiently ablating cancerous cells via the photothermal effect.

3.5 In vivo photothermal therapy

Finally, we investigated the in vivo antitumor PTT effectiveness of free NGO and UCNP@NGO on the tumors of mice. The mice were randomly divided into six groups with tumors of about 100 mm3: (a) PBS only, (b) PBS + laser, (c) free NGO only, (d) free NGO + laser, (e) UCNP@NGO only, and (f) UCNP@NGO + laser. All the groups were intratumorally injected, and the mice of groups (a), (c), and (e) were injected without laser irradiation, while the mice of groups (b), (d), and (f) were injected with the corresponding agents and then irradiated with the 808 nm laser (1.0 W cm−2) for 5 min. During irradiation, the tumor temperature at different time points was monitored with an IR thermal camera. High-contrast real-time IR thermal imaging of the treated tumors was observed in vivo (Fig. 5(a)). In contrast, for the mice injected with PBS only, the tumor temperature was barely changed (∼6 °C) during the whole irradiation process. For the groups treated with the free NGO and UCNP@NGO, the intensity of the IR thermal signal of the tumor site increased greatly upon extended irradiation. Remarkably, the IR thermal signal of UCNP@NGO is more visible compared with that of free NGO, demonstrating the higher photothermal conversion efficiency of the UCNP@NGO at the tumor sites after injection. To quantify the differences between free NGO and UCNP@NGO, the tumor temperatures were also recorded. The temperature of the tumor injected with UCNP@NGO rapidly increased to ∼63 °C after 5 min (Fig. 5(b)), which is 10 °C higher than that of free NGO.
image file: c7bm01113j-f5.tif
Fig. 5 In vivo PTT. (a) IR-T images of tumor-bearing mice, tumors were injected with PBS, free NGO and UCNP@NGO with 808 nm laser irradiation. (b) Tumor temperature monitored with an IR thermal camera during irradiation. (c)Weight of the survival mice and (d) tumor growth curves after various treatments as indicated. (e) Average tumor weights collected from the mice at the end of the experiment. The error bars correspond to the mean ± standard deviations. (f) Representative photos of mice tumors from different groups at the end of the treatments.

Without NIR irradiation, the tumors treated with free NGO and UCNP@NGO grew rapidly, and showed no difference with the ones injected with PBS buffer in cancer therapy. This suggested that either laser irradiation or the free NGO, UCNP@NGO injection alone were not able to restrict tumor development (Fig. 5(d)). In comparison, the tumor growth in the group of mice treated with free NGO and UCNP@NGO under NIR irradiation was completely stopped at the end of the treatment, and even represented a negative growth in the size. The photographs and the weights of the isolated tumors confirmed that when the tumors were treated with free NGO and UCNP@NGO under NIR irradiation, the growth could be completely suppressed (Fig. 5(e) and (f)). Finally, during the therapeutic period, the body laser weight changes were minimal for all groups of mice, indicating that these agents had negligible systemic toxicity (Fig. 5(c)). Overall, the in vivo study demonstrated that the inhibition of autophagy efficiently enhanced the efficacy of PTT. Our results reveal that UCNP@NGO can act as a promising agent for the in vivo photothermal therapy of tumors.

4. Conclusions

In this work, multifunctional nanoparticles UCNP@NGO were prepared as a theranostic platform for UCL imaging and photothermal therapy of cancer. The photothermal conversion efficiency and stability of UCNP@NGO is much higher than that of free NGO. Excellent biocompatibility and low toxicity of the novel UCNP@NGO nanoplatform were demonstrated by cytotoxicity assays, and the nanocomposite showed great potential to serve as a UCL imaging probe of whole-body animals with high contrast for diagnosis. In addition, PTT treatments of UCNP@NGO on tumor cells both in vitro and in vivo indicated prominent performances of the nanoparticles in inhibiting tumor growth. Compared with UCNPs or free NGO alone, the combined UCNP and NGO showed a synergistic effect, resulting in a higher therapeutic efficacy for in vitro cancer therapy. These results highlight that the integration of these functionalities endows UCNP@NGO with remarkable abilities for cancer theranostics.

Conflicts of interest

There are no conflicts to declare.


This work was supported by the National Key Research and Development Program of China (No. 2016YFA0201400), the NSFC program of China (51574267), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_14R58), the National Natural Science Youth Foundation of China (No. 21204051), the Beijing Education Committee Foundation program, and the Technology Foundation for Selected Overseas Chinese Scholar, Ministry of Personnel of China.

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Electronic supplementary information (ESI) available: TEM images, DLS, up-conversion luminescence spectra, FTIR and TGA of UCNP@OA and UCNP@NGO. The PTT of NGO and UCNP@NGO. See DOI: 10.1039/c7bm01113j

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