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photodynamic cancer therapy
Biomaterials 34 (2013) 7715e7724
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Graphene oxide covalently grafted upconversion nanoparticles for combined NIR mediated imaging and photothermal/photodynamic cancer therapy Yinghui Wang, Hengguo Wang, Dapeng Liu, Shuyan Song, Xiao Wang, Hongjie Zhang* State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry (CIAC), Chinese Academy of Sciences (CAS), Changchun 130022, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 April 2013 Accepted 23 June 2013 Available online 13 July 2013
Theranostics, the integration of diagnostics and therapies, has become a new concept in the battles with various major diseases such as cancer. Here, we report a multifunctional nanoplatform, which is developed by covalently grafting coreeshell structured upconversion nanoparticles (UCNPs) with nanographene oxide (NGO) via bifunctional polyethylene glycol (PEG), and then loading phthalocyanine (ZnPc) on the surface of NGO. The obtained UCNPs-NGO/ZnPc nanocomposites are not only be used as upconversion luminescence (UCL) imaging probes of cells and whole-body animals with high contrast for diagnosis, but also can generate cytotoxic singlet oxygen under light excitation for photodynamic therapy (PDT), as well as rapidly and efficiently convert the 808 nm laser energy into thermal energy for photothermal therapy (PTT). A remarkably improved and synergistic therapeutic effect compared to PTT or PDT alone is obtained, providing high therapeutic efficiency for cancer treatment. Therefore, benefiting from the unique multifunctional hybrid nanostructure, UCNPs-NGO/ZnPc nanocomposites developed herein are promising as an integrated theranostic probe for potential UCL image-guided combinatorial PDT/PTT of cancer. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Multifuntional nanoplatform Upconversion luminescence imaging Photothermal therapy Photodynamic therapy Synergistic effect
1. Introduction The growing demand for advancement in cancer diagnosis and therapy has triggered significant research efforts to construct theranostics nanoplatforms integrating imaging and therapy into a single system for imaging-guided, visualized cancer therapy due to the higher therapeutic efficiency and reduced side effects [1,2]. Imaging probes, as the tool to identify the location of cancer cell, monitor the biodistribution of nanocomposites and assess the therapeutic efficacy, are one of the most important parts of the theranostic nanoplatform. Recently, upconversion nanoparticles (UCNPs), particularly lanthanide-doped rare-earth nanocrystals, have been proposed as new generation of fluorescent probes with great potential in biomedical imaging since they have shown several significant advantages such as a sharp emission bandwidth, long lifetime, tunable emission, high photostability, and low cytotoxicity [3e6]. More importantly, UCNPs utilize near infrared (NIR) excitation within the “optical transmission window” of biological
* Corresponding author. Tel.: þ86 431 85262127; fax: þ86 431 85698041. E-mail address: [email protected] (H. Zhang). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.06.045
tissues (700e1000 nm), thereby significantly enhancing penetration depths and minimizing background autofluorescence, photobleaching as well as photodamage to biological specimens [7e11]. Benefiting from such unique advantages, UCNPs have been successfully employed as promising contrast agents for in vitro cell imaging and in vivo whole-animal imaging [3,6,12e24]. Lately, the construction of theranostics nanoplatform composed of UCNPs and various therapeutic agents, such as anti-cancer drugs [25], photosensitizers (PS) [26,27] and gold nanostructures [28,29], for potential therapeutic applications, especially noninvasive photodynamic therapy (PDT) and photothermal therapy (PTT) has been a research hotspot in the forefront of materials science. However, there exist several scientific and technical challenges of UCNPs-based theranostics nanoplatform for application in the clinic. First, the therapeutic effect of UCNPs-based PDT is unsatisfactory owing to the low upconversion luminescence (UCL) emission quantum yield (less than 1%) and limited resonance energy transfer efficiency [30]. Second, the strategies of direct combination of photothermal agent to UCNPs also need more investigation because the photothermal agents often quench the emission of the UCNPs. For example, coating gold nanoshells on the surface of UCNPs has been reported that it greatly suppressed the emission
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due to the strong scattering of the excitation irradiation [28,29]. Finally, nanocarriers used for constructing multifunctional nanoplatform, including liposomes, polymers, micelles, mesoporous silica etc. [31], are only the container of the incorporated therapeutic agents. In this respect, they themselves don’t own any therapeutic function, resulting in sophisticated preparation process to assemble multiple treatment modalities nanoplatform. Over the years, extensive efforts have been devoted to addressing the first challenge, by developing coreeshell structured UCNPs [32,33] and grafting PS on the surface of UCNPs [34], in an attempt to enhance the UCL emissions and improve the resonance energy transfer efficiency. However, insufficient emphasis has been placed on dealing with the latter two challenges. Along this line, if nanographene oxide (NGO), which can be used as both carrier for PS and photothermal agent for PTT due to its large surface area and intrinsic high NIR absorbance [35e41], is combined with coreeshell structured UCNPs via long-chain joint molecule, the resulting theranostic nanoplatform will exhibit both high fluorescent intensity for imaging and improved therapeutic effect owing to the combination of PDT and PTT. Building from these ideas, herein we design and develop a multifunctional nanocomposite, which is synthesized by covalently grafting coreeshell structured UCNPs with NGO via bifunctional polyethylene glycol (PEG), and then loading phthalocyanine (ZnPc) on the surface of NGO. Benefiting from the unique hybrid nanostructure, this nanocomposite integrates the multifunctions of PDT, PTT and UCL imaging into a single nanoplatform, which could act as an integrated theranostic probe for UCL image-guided combinatorial PDT/PTT of cancer (Scheme 1). Remarkably, both imaging and dual-mode treatments in this nanoplatform are stimulated by light, which exhibits remarkable advantages in terms of enhancing cancer killing specificity and reducing side effects since only the tumor lesion exposed to the light, in comparison to conventional cancer therapies. Moreover, a synergistic effect of combined noninvasive photodynamic and photothermal therapy is expected to improve the therapeutic efficiency, decrease the dosage-limiting toxicity and tissue damage by over-heating. 2. Materials and methods 2.1. Materials Rare-earth oxides RE2O3 (99.99%) (RE ¼ Y, Yb, Er, Tm) were purchased from Science and Technology Parent Company of Changchun Institute of Applied Chemistry. Oleic acid (>90%), 1-octadecene (ODE; >90%), 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC), poly(allylamine) and zinc phthalocyanine were purchased from Aldrich. Amino carboxylic PEG was purchased from Sunbio Inc. All above chemicals were used directly without further purification. Rare earth chloride (RECl3) stock solutions of 0.2 M were obtained by dissolving the corresponding metal oxide in hydrochloric acid under heating with agitation. 2.2. Synthesis of therapeutic probe UCNPs-NGO/ZnPc 2.2.1. Synthesis of coreeshell NaYF4: Yb3þ, Er3þ, Tm3þ/NaYF4 UCNPs Hexagonal-phase NaYF4: Yb3þ, Er3þ, Tm3þ core nanoparticles were synthesized following previously reported method [42e44]. In details, 2 mL solution of RECl3 (0.2 M, RE ¼ Y, Yb, Er, Tm) in methanol was added to a 50 mL flask containing 3 mL oleic acid and 7 mL 1-octadecene. The solution was heated to 110 C for 30 min with vigorous magnetic stirring to remove water and oxygen under argon flow protection, and then heated to 160 C for 30 min. After the solution was cooled to room temperature, a 5 mL methanol solution of NH4F (1.6 mmol) and NaOH (1 mmol) was added, and then the solution was kept at 50 C for 30 min. After methanol was evaporated, the solution was heated to 300 C under an argon atmosphere for 1.5 h and then cooled to room temperature. The nanocrystals were precipitated by addition of ethanol, collected after centrifugation, and washed with ethanol for several times, and dispersed in cyclohexane. The NaYF4 shell precursor was prepared by mixing 2 mL methanol solution of YCl3 (0.2 M), 3 mL oleic acid and 7 mL 1-octadecene, and the solution was heated to 110 C for 30 min, and then to 160 C for 30 min. Next, the solution was cooled to 50 C. The core nanoparticles in 4 mL cyclohexane were added along with 5 mL methanol solution of NH4F (1.6 mmol) and NaOH (1 mmol), and the solution was
stirred at 50 C for 30 min. After the removal of cyclohexane and methanol, the solution was heated to 300 C under argon for 1.5 h and cooled to room temperature. The nanocrystals were collected after centrifugation. 2.2.2. Synthesis of amino-functionalized coreeshell NaYF4: Yb3þ, Er3þ, Tm3þ/NaYF4 UCNPs In order to improve the efficiency of phase transfer, we first synthesized the ligand-free UCNPs following the pervious report [45]. The obtained coreeshell nanoparticles were dispersed in a 10 mL aqueous solution, and stirred for 12 h while maintaining the pH at 4 by adding a solution of HCl (0.1 M). Then the solution was extracted with water diethyl ether three times to remove the oleic acid. The ligandfree UCNPs in the water were obtained by centrifugation after precipitation with acetone. Finally the particles were dispersed in water. Subsequently, 0.1 mL poly(allylamine) 20% solution in water was dispersed in 10 mL water, then the ligand-free UCNPs were added dropwise into poly(allylamine) solution and stirred vigorously over 48 h at room temperature. After centrifugation, UCNPs-NH2 was redispersed in water, and the solution was stable for several days without obvious aggregation. 2.2.3. Synthesis of PEG-functionalized NGO GO was prepared following a modified Hummers method [46,47]. NaOH (0.12 g/ mL) was added to 10 mL GO aqueous suspension (w3 mg/mL), and sonicated for about 4 h. The resulting solution was neutralized, purified by repeated rinsing and centrifugation. The obtained GOeCOOH (1 mg/mL) solution was then mixed with an aqueous solution of amino carboxylic PEG (3 mg/mL). After sonicate for 5 min, EDC was then added to the mixture in two equal portions to give a final concentration of 1 mg/mL totally. The resulting solution was stirred at room temperature for 24 h and then centrifuged filtration through Amicon centrifugal filters (Millipore) with 5 kDa and washed with water to remove the amino carboxylic PEG. 2.2.4. Synthesis of UCNPs-NGO NGO-PEG (0.5 mg/mL) was mixed with a 1 mL aqueous solution of EDC (0.03 mmol), then sonicated for 30 min at room temperature. Subsequently, UCNPsNH2 (0.5 mg/mL) solution was added dropwise into above mixture and stirred vigorously for 24 h. UCNPs-NGO composites were centrifuged and washed with water. 2.2.5. Synthesis of UCNPs-NGO/ZnPc ZnPc (10 mg) was dissolved in 100 mL DMF. After leaving for 24 h, the supernatant was decanted. UCNPs-NGO (5 mg) was dispersed by sonication in 10 mL ZnPc DMF solution. After stirring for 48 h, UCNPs-NGO/ZnPc was obtained by centrifugation, and washed with ethanol to remove excess ZnPc. The resulting UCNPs-NGO/ ZnPc was freeze-dried. 2.3. Detection of singlet oxygen The generation of singlet oxygen was detected by the chemical probe DPBF. In a typical experiment, 20 mL DPBF solution (10 mM) was added to 2 mL UCNPs-NGO/ ZnPc solution, and then transferred into a 10 mm cuvette. The mixture was irradiated with a 630 nm laser at a power density of 50 mW cm2 for 10 min, and the absorption intensity of DPBF at 410 nm was recorded every minute. For comparison, the absorbance of free ZnPc and DPBF mixture was also recorded at the same conditions. 2.4. Cytotoxicity assay The human nasopharyngeal epidermal carcinoma KB cell and human cervix cancer cell line HeLa cell, provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China), were cultured in RPMI-1640 culture medium containing 10% FBS and 1% penicillin/streptomycin at 37 C under 5% CO2. The cells were seeded in 96well plates (5 104 cells per well) for 24 h, and then different concentrations of UCNPs-NGO (0, 20, 40, 80, 160 and 320 mg/mL, diluted in RPMI 1640) were added to the wells. After incubation for 24 h, 10 mL MTT solution (5 mg/mL MTT in PBS, pH 7.4) was added to each well and the plate was incubated for an additional 4 h. After removing the medium, the wells were washed by PBS, and then the intracellular formazan crystals were extracted into 100 mL DMSO. The absorbance was recorded at 490 nm by a plate reader, and the cell viability could be calculated from the average value of six parallel wells. 2.5. PDT and PTT treatments Hela cells were precultured in 96-well plates at 5 104 cells per well for 24 h and then added UCNPs-NGO/ZnPc at different concentrations from 0 to 320 mg/mL. After 24 h of incubation, the cells were washed twice with PBS and irradiated using 808 nm NIR laser at a power density of 2 W cm2 for 10 min. Then, the cells were allowed to incubate for another 24 h. Thereafter, the standard MTT assay was carried out to determine the cell viabilities. The method of detecting the effect of PDT treatment was the same as that used in PTT treatments, except that we used 630 nm laser at a power density of 50 mW cm2 for irradiation.
Y. Wang et al. / Biomaterials 34 (2013) 7715e7724 Scheme 1. (a) Schematic illustration of the synthetic procedure for UCNPs-NGO: Numbers of coreeshell structured UCNPs being covalently grafted with NGO via bifunctional polyethylene glycol; (b) Schematic illustration of UCNPsNGO/ZnPc as a multifunctional theranostic nanoplatform for cancer treatment. 7717
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Y. Wang et al. / Biomaterials 34 (2013) 7715e7724 2.8. AO/EB staining of HeLa cells After cultured with 80 mg/mL UCNPs-NGO/ZnPc nanocomposites for 24 h, the cells (control and experimental groups) were treated without or with single or combinational laser. After another 24 h incubation, 10 mL of 50 mg/mL AO and EB mixed dye solution in PBS was added to each Petri dish and co-cultured with cells in darkness for 20 min at room temperature. Thereafter, the cells were washed with PBS to remove excess dye and detached cells. Fluorescence microscope was used to monitor the cell morphology. AO emissions were measured in the green channel when excited by 488 nm, and EB emissions were collected in the red channel when excited by 543 nm.
3. Results and discussion 3.1. Preparation and characterization
Fig. 1. TEM image of monodisperse UCNPs-NH2 nanoparticles in water.
For synergistically enhanced anti-cancer effect of the combination of PDT and PTT, Hela cells were seeded in 96-well plates at 5 104 cells per well for 24 h, and then 40 or 80 mg/mL UCNPs-NGO/ZnPc nanocomposites were added. After 24 h of incubation, the cells were exposed to the 808 nm NIR laser at a power density of 2 W cm2 for 10 min. Subsequently, cells were irradiated by 630 nm laser at a power density of 50 mW cm2 for 10 min. After another 24 h of incubation, cell viability was measured according to the standard MTT assay method. 2.6. UCL imaging of living cells For confocal imaging, KB cells and HeLa cells were plated on 14 mm glass coverslips under 100% humidity and allowed to adhere for 24 h. The cells were stained with 200 mg/mL UCNPs-NGO at 37 C for 2 h. Prior to imaging, the coverslips were washed twice with PBS in order to remove excess UCNPs-NGO. Confocal imaging of cells was performed with a modified Olympus FV1000 laser scanning upconversion luminescence microscope (LSUCLM) equipped with a continuous-wave (CW) laser at 980 nm (Connet Fiber Optics, China). Upconversion luminescence signals were detected in the green channel (520e560 nm) and red channel (600e700 nm). 2.7. UCL imaging in vivo of mice All animal studies were performed in accordance with institutional and national guidelines. Kunming mouse (18 g) was anesthetized (with 10% chloral hydrate 100 L) and was subcutaneously injected with the UCNPs-NGO. Subsequently, the mouse was imaged by a modified in vivo Maestro whole-body imaging system with an external 980 nm laser as the excitation source.
For highly efficient UCL, coreeshell structured Tm3þ/Er3þ/Yb3þ co-doped NaYF4@NaYF4 nanocrystals were synthesized as fluorescent probe using previously reported method [42e44]. The average diameters of the core and coreeshell UCNPs were determined to be 28 nm and 40 nm by transmission electron microscopy (TEM), respectively, as presented in Fig. S1 (Supporting Information). The X-ray diffraction (XRD) pattern showed that the nanoparticles were hexagonal b-NaYF4 (Fig. S2). The UCNPs were further converted into hydrophilic UCNPs-NH2 via a ligand exchanging process using poly(allylamine) (PAAm) as surface coating agent to replace the original oleic acid ligands. The TEM image (Fig. 1) demonstrated high monodispersity of UCNPs-NH2 in water, which was ideal for biological applications. In order to endow the obtained UCNPs with multimodal therapeutic functions, NGO as both PTT agent and carriers for PS was covalently grafted with UCNPs via a carbodiimide cross-linking reaction between the amino group of UCNPs-NH2 and the carboxyl group of NGO-PEG following the protocol as illustrated in Scheme 1a. NGO-PEG was obtained by covalently grafting amino carboxylic PEG onto NGO sheet, and the left carboxylic groups at the PEG terminals were available for fluorescent probes. The successful PEG functionalization was evidenced by Fourier-transform infrared spectroscopy (FT-IR; Fig. S3). The morphology of NGOPEG was characterized by atomic force microscope (AFM, Fig. 2a). NGO-PEG was almost single layered sheet with average topographic height of w1.2 nm, as shown in Fig. 2b. To verify the potential of using NGO in PTT, we detected the temperature variation of NGO-PEG solution exposed to an 808 nm NIR laser at a power density of 2 W/cm2 using a thermal imaging camera (Fig. 3). Before NIR exposure, the maximum temperature was about 23 C, but the temperature of NGO-PEG solution increased rapidly to 58 C within 3 min after exposing to NIR light (Fig. S4), which indicated that the NGO could rapidly and efficiently convert the 808 nm laser energy
Fig. 2. (a) AFM image of NGO-PEG deposited on mica substrate, and (b) the height profile of the AFM image, showing NGO-PEG was almost single layered sheet.
Y. Wang et al. / Biomaterials 34 (2013) 7715e7724 Fig. 3. Temperature change images of the NGO-PEG solution exposed to the 808 nm laser for different time periods at a power density of 2 W/cm2, showing NGO could rapidly and efficiently convert the 808 nm laser energy into thermal energy.
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Fig. 4. (a) Upconversion emission spectra of UCNPs-NH2 and UCNPs-NGO excited with a CW 980 nm laser. (b) Photographs of UCNPs-NGO under ambient light (left) and under CW 980 nm laser irradiation (right).
Fig. 5. Time-course generation of singlet oxygen by UCNPs-NGO/ZnPc and free ZnPc detecting by the bleaching of DPBF absorption at 410 nm under 630 nm laser irradiation.
into thermal energy. The reason for selecting PEG as linker was twofold. On one hand, PEG has good compatibility with biological system, which renders NGO high aqueous solubility and stability in physiological solutions including serum [48]. On the other hand, bonding UCNPs at the end of the long chain of PEG molecules can reduce the fluorescence quenching by NGO. The emission spectra of UCNPs-NH2 and UCNPs-NGO (Fig. 4a) displayed characteristic green, red (in web version) and NIR emission bands under CW excitation at 980 nm. The visible emission bands could be assigned to the 2H11/2 / 4I15/2, 4S3/2 / 4I15/2 and 4F9/2 / 4I15/2 transition of Er3þ ions, respectively. The NIR emission band around 808 nm was attributed to Tm3þ transitions from 3H4 to 3H6, which was more suitable for in vivo imaging owing to the higher penetration depth. Although the emission intensity of UCNPs-NGO was a little weaker than that of UCNPs-NH2 owing to the part absorption by NGO, the retained emission was still strong enough to be observed by naked eye (Fig. 4b). This result demonstrates that our strategy is highly effective to avoid quenching UCL by therapeutic agent, endowing the nanoplatform with both high fluorescent intensity and high therapeutic effect. To achieve multimodal efficient therapy, aromatic ZnPc with high optical absorption coefficient in the phototherapeutic window of 600e800 nm was used as a model PS, and loaded on the surface of UCNPs-NGO for PDT by mixing the ZnPc/DMF solution with the UCNPs-NGO aqueous solution for 48 h. The UVevis spectra of UCNPs-NGO/ZnPc showed the absorption bands were red shifted and broadened compared with those of ZnPc dissolved in DMF (Fig. S5). Such result manifested a strong interaction of ZnPc with UCNPs-NGO via pep stacking. Following the method reported by
Shi’s group [49], the loading efficiency was calculated to be 11%. Such high PS loading efficiency indicated that UCNPs-NGO was a promising PS nanocarrier for PDT. Singlet oxygen generation was the critical step in PDT, so the 1O2 generation ability of UCNPs-NGO/ ZnPc was a very important index to illustrate its application potential in PDT. We monitored the singlet oxygen production of UCNPs-NGO/ZnPc and free ZnPc by the chemical probe 1,3-
Fig. 6. Cell viability of HeLa and KB cells incubated with UCNPs-NGO at different concentration for 24 h, respectively, showing the low cytotoxicity of UCNPs-NGO.
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Fig. 7. Upconversion luminescence imaging at green channel (lem ¼ 520e560 nm) and red channel (lem ¼ 600e700 nm) of HeLa and KB cells stained with 200 mg/mL UCNPs-NGO for 2 h at 37 C. Overlay of luminescence image and bright field image were also shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
diphenylisobenzofuran (DPBF), whose absorbance at 410 nm would be diminished in the presence of 1O2. Fig. 5 showed the absorbance of solutions of DPBF and UCNPs-NGO/ZnPc or free ZnPc as a function of exposure time to a 630 nm laser irradiation. The absorbance intensity of DPBF displayed a continuous decrease with 10 min for both UCNPs-NGO/ZnPc and free ZnPc. Although the 1O2 production ability by UCNPs-NGO/ZnPc was weaker than that of free ZnPc owing to the quenching effect by NGO, it still retained about 60% of free ZnPc, which allowed us to use UCNPs-NGO/ZnPc for PDT treatment of cancer cells. 3.2. In vitro cytotoxicity Prior to the theranostic application of UCNPs-NGO nanoplatform, the in vitro cytotoxicity of UCNPs-NGO was assessed using a traditional MTT assay. Fig. 6 showed the in vitro cell viability of human cervix cancer HeLa cell and human nasopharyngeal epidermal cancer KB cell incubated with UCNPs-NGO with different concentration ranging from 20 to 320 mg/mL for 24 h. The cell viabilities were higher than 90% for both HeLa and KB cell, even at the high concentration of 320 mg/mL. These data show satisfactory results for in vitro non-cytotoxicity for all dosages of UCNPs-NGO. Good biocompatibility and low cytotoxicity imply that UCNPsNGO can serve as a theranostic probe for simultaneous UCL imaging and combinatorial PDT/PTT of cancer.
3.3. Upconversion luminescence (UCL) imaging in vitro and in vivo Cellular uptake of UCNPs-NGO was investigated by laser scanning upconversion luminescence microscopy (LSUCLM). After incubation with 200 mg/mL UCNPs-NGO for 2 h at 37 C, an intense UCL signal from 520 to 560 nm and 600e700 nm was observed from the HeLa cells (Fig. 7) under CW excitation at 980 nm. The nanocomposites were mainly located in cytoplasm and perinuclear regions, which indicated that UCNPs-NGO penetrated the cell membrane of living HeLa cells. To further verify whether UCNPsNGO can be used for other cancer cells, KB cells were chosen to uptake the nanocomposites. From the confocal luminescence imaging of KB cells, we can see that UCNPs-NGO has crossed the cell membrane and entered the cytoplasm, which is similar to that of HeLa cells. The above results demonstrated that UCNPs-NGO is promising candidate for cancer cellular labeling and imaging. Encouraged by the results of UCL imaging of cancer cells in vitro, we subcutaneously injected UCNPs-NGO onto the back of a white Kunming mouse, which was then imaged by a modified in vivo Maestro whole-body imaging system with an external 980 nm laser as the excitation source. After injection, a strong UCL signal arose from the place where the nanocomposites were injected (Fig. 8), which revealed that the UCL signal could easily penetrate these tissues of the mouse. More importantly, almost no autofluorescence was detected elsewhere. The result confirms the
Fig. 8. In vivo upconversion luminescence imaging of a white Kunming mouse after subcutaneous injection 200 mg/mL UCNPs-NGO.
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Fig. 9. (a) In vitro photodynamic and photothermal treatment of cancer cell: viability of HeLa cells treated with UCNPs-NGO/ZnPc of different concentrations without and with irradiation by 808 nm or 630 nm laser. (b) In vitro combined photodynamic and photothermal treatment of cancer cell: viability of HeLa cells treated with UCNPs-NGO/ZnPc at 40 mg/mL and 80 mg/mL. Black, red, blue, and green bars represent cells without any light irradiation, with only 808 nm laser irradiation, with only 630 nm laser irradiation, and with both 808 nm and 630 nm laser irradiation, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
capability of UCNPs-NGO nanocomposites for high contrast UCL imaging in vivo. 3.4. Photodynamic therapy and photothermal therapy Cell destruction by both PDT and PTT was studied using HeLa cells. Cells were incubated with UCNPs-NGO/ZnPc at a series of concentrations for 24 h and then irradiated with either 630 nm or 808 nm laser for 10 min. In a concentration-dependent manner, without light exposure, UCNPs-NGO/ZnPc exhibited negligible dark toxicity to HeLa cells at concentrations up to 320 mg/mL (Fig. 9a). As indicated by the 1O2 generation ability studies, singlet oxygen was produced by irradiating of UCNPs-NGO/ZnPc with 630 nm laser at a density of 50 mW/cm2. Therefore, when HeLa cells were exposed to laser, the cell viability decreased significantly with the increase of the concentration of UCNPs-NGO/ZnPc, manifesting the feasibility of the nanocomposites to destroy the cancer cells by 1O2. As for the effect of PTT, the cell viability for UCNPs-NGO/ZnPc at the concentration from 20 to 320 mg/mL decreased from about 95% to 30% after irradiating for 10 min with 808 nm laser, demonstrating an obvious photothermal efficacy. These results clearly demonstrate that UCNPs-NGO/ZnPc has a high possibility for PDT and PTT. A cell viability assay was conducted at two UCNPs-NGO/ZnPc concentrations (40 and 80 mg/mL, denoted as group A and B) (Fig. 9b) in order to further quantify the synergistically enhanced anti-cancer effect of the combination of the two treatment approaches. The control cells, which were incubated with different concentration of UCNPs-NGO/ZnPc without any light irradiation, showed nearly no damage. After irradiation of 630 nm laser, the cell viability of group A and B decreased to about 75% and 50%, respectively. Because the power and optical dose of the 630 nm light applied in this experiment were extremely low without causing any noticeable heating of UCNPs-NGO/ZnPc, cell mortality only induced by singlet oxygen generation upon excitation of the photosensitizer. In the same time, NIR laser irradiation decreased the cell viability to about 85% and 75% for the two groups based on the photothermal killing of cancer cells. As expected, when the two treatments were combined, the cell viability was remarkably reduced to 35% and 15%, respectively, which is evidently much lower than that by the individual ones. The remarkably improved therapeutic effect may be attributed to the photothermal effect of graphene that not only can “cook” the cancer cells but also enhance the delivery of PDT agents for improved photodynamic cancer cell
killing [50]. Therefore, UCNPs-NGO/ZnPc can destroy cancer cells more efficiently when used for combined PDT and PTT. Taken together, the nanocomposites as-assembled for an adaptive tumor theranostic platform could be proposed as follows. Preliminarily, UCNPs-NGO/ZnPc would be localized in tumor tissues owing to the enhanced permeability and retention effect and further internalized by tumor cell through the energy-dependent endocytosis process, followed by the formation of endosomes. Meanwhile, the tumor cells could be clearly observed in the UCL image with a high signal-to-background ratio. Then, selectively destroying the tumor cells guided by UCL imaging was carried out in a noninvasive manner using laser irradiation, which minimized damage of the surrounding normal cells. Finally, synergistically enhanced anticancer efficacy of combinatorial PDT/PTT was achieved. Furthermore, for the sake of visualizing the tumorigenic cell ablation efficacy and demonstrating the relevant mechanism in vitro, HeLa cells were incubated with UCNPs-NGO/ZnPc (80 mg/ mL) for 12 h. After a series of treatment with single or combinational laser, the samples were stained with a mixture of the nucleic acid-specific dyes acridine orange (AO) and ethidium bromide (EB) following the standard staining protocols and immediately observed by fluorescence microscopy. It is known that AO can enter live cells and give the nuclei a green fluorescence, while EB is excluded by intact tumor cell membranes but stains the nuclei of dead cells fluorescent orange. So we could assess the cell viability by observing fluorescent microscopic image. In the control experiment, cell viability exhibits no obvious decline, indicating the nanocomposite itself could not lead to cell death without irradiation (Fig. 10aeb). However, it is observed that several cells show a bright orange red color (in web version) after irradiation with 808 nm laser (Fig. 10ced). The damage of cells may be caused by localized hyperthermia. After irradiation of 630 nm laser, the cell viability distinctively decreases, and nearly 40% cell nucleus displays orange red light (in web version) (Fig. 10eef), showing late apoptotic or necrotic character. Such result demonstrates that UCNPs-NGO/ZnPc nanocomposite could kill cells through both photodynamic and photothermal effects under laser irradiation. After combinational treatment with the two lasers, as presented in Fig. 10geh, the necrotic areas appear, and none of the rest cells is alive. This further certified that the combined PDT and PTT multimodal nanoplatform based on UCNPs-NGO/ZnPc nanocomposites was a more efficient therapeutic regimen against cancer cells than either PDT or PTT alone.
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Fig. 10. Representative fluorescent microscopic images of HeLa cells treated with 80 mg/mL UCNPs-NGO/ZnPc with or without single or combinational laser illumination, and then the cells were stained by nucleic acid-specific dyes AO and EB. (aeb) without irradiation; (ced) under irradiation of 808 nm laser; (eef) under irradiation of 630 nm laser; (geh) under combinational laser irradiation.
4. Conclusions We have prepared UCNPs-NGO/ZnPc as a theranostic platform for UCL image-guided combinatorial PDT/PTT of cancer. Cytotoxicity assays demonstrated good biocompatibility and low toxicity of the UCNPs-NGO/ZnPc. The nanocomposite could be used as UCL imaging probe of cells and whole-body animals with high contrast for diagnosis. At the same time, significantly greater cell killing was obtained because UCNPs-NGO/ZnPc was not only excellent nanocarrier to load ZnPc for PDT, also could kill cells by a photothermal effect. Compared with photodynamic or photothermal treatment
alone, the combined treatment showed a synergistic effect, resulting in higher therapeutic efficacy for in vitro cancer therapy. These results highlight that integration of these functionalities endows UCNPs-NGO/ZnPc with the potential promise for cancer theranostics. Acknowledgments The authors are grateful to the financial aid from the National Natural Science Foundation of China (Grant Nos. 21001101, 21071140, 91122030 and 21210001), ‘863’-National High
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Technology Research and Development Program of China (Grant No. 2011AA03A407) and National Natural Science Foundation for Creative Research Group (Grant No. 21221061). We wish to thank Prof. F. Y. Li (FU) for kindly providing UCL imaging measurements. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2013.06.045. References [1] Greco F, Vicent M. Combination therapy: opportunities and challenges for polymer-drug conjugates as anticancer nanomedicines. Adv Drug Deliv Rev 2009;61:1203e13. [2] Lane D. Designer combination therapy for cancer. Nat Biotechnol 2006;24: 163e4. [3] Wu S, Han G, Milliron DJ, Aloni S, Altoe V, Talapin DV, et al. Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals. Proc Natl Acad Sci U S A 2009;106:10917e21. [4] Park YI, Kim JH, Lee KT, Jeon KS, BinNa H, Yu JH, et al. Nonblinking and nonbleaching upconverting nanoparticles as an optical imaging nanoprobe and T1 magnetic resonance imaging contrast agent. Adv Mater 2009;21: 4467e71. [5] Li Z, Zhang Y, Jiang S. Multicolor core/shell-structured upconversion fluorescent nanoparticles. Adv Mater 2008;20:4765e9. [6] Liu Q, Sun Y, Yang T, Feng W, Li C, Li F. Sub-10 nm hexagonal lanthanidedoped NaLuF4 upconversion nanocrystals for sensitive bioimaging in vivo. J Am Chem Soc 2011;133:17122e5. [7] Auzel F. Upconversion and anti-stokes processes with f and d ions in solids. Chem Rev 2004;104:139e74. [8] Wang F, Liu XG. Recent advances in the chemistry of lanthanide-doped upconversion nanocrystals. Chem Soc Rev 2009;38:976e89. [9] Wang F, Banerjee D, Liu YS, Chen XY, Liu XG. Upconversion nanoparticles in biological labeling, imaging, and therapy. Analyst 2010;135:1839e54. [10] Haase M, Schäfer H. Upconverting nanoparticles. Angew Chem Int Ed 2011;50:5808e29. [11] Zhou J, Liu Z, Li FY. Upconversion nanophosphors for small-animal imaging. Chem Soc Rev 2012;41:1323e49. [12] Nyk M, Kumar R, Ohulchanskyy TY, Bergey EJ, Prasad PN. High contrast in vitro and in vivo photoluminescence bioimaging using near infrared to near infrared upconversion in Tm3þ and Yb3þ doped fluoride nanophosphors. Nano Lett 2008;8:3834e8. [13] Jalil RA, Zhang Y. Biocompatibility of silica coated NaYF(4) upconversion fluorescent nanocrystals. Biomaterials 2008;29:4122e8. [14] Hu H, Yu M, Li F, Chen Z, Gao X, Xiong L, et al. Facile epoxidation strategy for producing amphiphilic up-converting rare-earth nanophosphors as biological labels. Chem Mater 2008;20:7003e9. [15] Idris NM, Li ZQ, Ye L, Sim EKW, Mahendran R, Ho PCL, et al. Tracking transplanted cells in live animal using upconversion fluorescent nanoparticles. Biomaterials 2009;30:5104e13. [16] Xiong LQ, Chen ZG, Yu MX, Li FY, Liu C, Huang CH. Synthesis, characterization, and in vivo targeted imaging of amine-functionalized rare-earth upconverting nanophosphors. Biomaterials 2009;30:5592e600. [17] Xiong LQ, Chen ZG, Tian QW, Cao TY, Xu CJ, Li FY. High contrast upconversion luminescence targeted imaging in vivo using peptide-labeled nanophosphors. Anal Chem 2009;81:8687e94. [18] Yu M, Li F, Chen Z, Hu H, Zhan C, Yang H, et al. Laser scanning up-conversion luminescence microscopy for imaging cells labeled with rare-earth nanophosphors. Anal Chem 2009;81:930e5. [19] Boyer JC, Manseau MP, Murray JI, van Veggel CJM. Surface modification of upconverting NaYF4 nanoparticles with PEG-phosphate ligands for NIR (800 nm) biolabeling within the biological window. Langmuir 2010;26: 1157e64. [20] Kobayashi H, Kosaka N, Ogawa M, Morgan NY, Smith PD, Murray CB, et al. In vivo multiple color lymphatic imaging using upconverting nanocrystals. J Mater Chem 2009;19:6481e4. [21] Wang M, Mi CC, Wang WX, Liu CH, Wu YF, Xu ZR, et al. Immunolabeling and NIR-excited fluorescent imaging of HeLa cells by using NaYF4:Yb, Er upconversion nanoparticles. ACS Nano 2009;3:1580e6. [22] Yu X, Li M, Xie M, Chen L, Li Y, Wang Q. Dopant-controlled synthesis of watersoluble hexagonal NaYF4 nanorods with efficient upconversion fluorescence for multicolor bioimaging. Nano Res 2010;3:51e60.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Graphene oxide covalently grafted upconversion nanoparticles for combined NIR mediated imaging and photothermal/photodynamic cancer therapy Yinghui Wang, Hengguo Wang, Dapeng Liu, Shuyan Song, Xiao Wang, Hongjie Zhang* State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry (CIAC), Chinese Academy of Sciences (CAS), Changchun 130022, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 April 2013 Accepted 23 June 2013 Available online 13 July 2013
Theranostics, the integration of diagnostics and therapies, has become a new concept in the battles with various major diseases such as cancer. Here, we report a multifunctional nanoplatform, which is developed by covalently grafting coreeshell structured upconversion nanoparticles (UCNPs) with nanographene oxide (NGO) via bifunctional polyethylene glycol (PEG), and then loading phthalocyanine (ZnPc) on the surface of NGO. The obtained UCNPs-NGO/ZnPc nanocomposites are not only be used as upconversion luminescence (UCL) imaging probes of cells and whole-body animals with high contrast for diagnosis, but also can generate cytotoxic singlet oxygen under light excitation for photodynamic therapy (PDT), as well as rapidly and efficiently convert the 808 nm laser energy into thermal energy for photothermal therapy (PTT). A remarkably improved and synergistic therapeutic effect compared to PTT or PDT alone is obtained, providing high therapeutic efficiency for cancer treatment. Therefore, benefiting from the unique multifunctional hybrid nanostructure, UCNPs-NGO/ZnPc nanocomposites developed herein are promising as an integrated theranostic probe for potential UCL image-guided combinatorial PDT/PTT of cancer. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Multifuntional nanoplatform Upconversion luminescence imaging Photothermal therapy Photodynamic therapy Synergistic effect
1. Introduction The growing demand for advancement in cancer diagnosis and therapy has triggered significant research efforts to construct theranostics nanoplatforms integrating imaging and therapy into a single system for imaging-guided, visualized cancer therapy due to the higher therapeutic efficiency and reduced side effects [1,2]. Imaging probes, as the tool to identify the location of cancer cell, monitor the biodistribution of nanocomposites and assess the therapeutic efficacy, are one of the most important parts of the theranostic nanoplatform. Recently, upconversion nanoparticles (UCNPs), particularly lanthanide-doped rare-earth nanocrystals, have been proposed as new generation of fluorescent probes with great potential in biomedical imaging since they have shown several significant advantages such as a sharp emission bandwidth, long lifetime, tunable emission, high photostability, and low cytotoxicity [3e6]. More importantly, UCNPs utilize near infrared (NIR) excitation within the “optical transmission window” of biological
* Corresponding author. Tel.: þ86 431 85262127; fax: þ86 431 85698041. E-mail address: [email protected] (H. Zhang). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.06.045
tissues (700e1000 nm), thereby significantly enhancing penetration depths and minimizing background autofluorescence, photobleaching as well as photodamage to biological specimens [7e11]. Benefiting from such unique advantages, UCNPs have been successfully employed as promising contrast agents for in vitro cell imaging and in vivo whole-animal imaging [3,6,12e24]. Lately, the construction of theranostics nanoplatform composed of UCNPs and various therapeutic agents, such as anti-cancer drugs [25], photosensitizers (PS) [26,27] and gold nanostructures [28,29], for potential therapeutic applications, especially noninvasive photodynamic therapy (PDT) and photothermal therapy (PTT) has been a research hotspot in the forefront of materials science. However, there exist several scientific and technical challenges of UCNPs-based theranostics nanoplatform for application in the clinic. First, the therapeutic effect of UCNPs-based PDT is unsatisfactory owing to the low upconversion luminescence (UCL) emission quantum yield (less than 1%) and limited resonance energy transfer efficiency [30]. Second, the strategies of direct combination of photothermal agent to UCNPs also need more investigation because the photothermal agents often quench the emission of the UCNPs. For example, coating gold nanoshells on the surface of UCNPs has been reported that it greatly suppressed the emission
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due to the strong scattering of the excitation irradiation [28,29]. Finally, nanocarriers used for constructing multifunctional nanoplatform, including liposomes, polymers, micelles, mesoporous silica etc. [31], are only the container of the incorporated therapeutic agents. In this respect, they themselves don’t own any therapeutic function, resulting in sophisticated preparation process to assemble multiple treatment modalities nanoplatform. Over the years, extensive efforts have been devoted to addressing the first challenge, by developing coreeshell structured UCNPs [32,33] and grafting PS on the surface of UCNPs [34], in an attempt to enhance the UCL emissions and improve the resonance energy transfer efficiency. However, insufficient emphasis has been placed on dealing with the latter two challenges. Along this line, if nanographene oxide (NGO), which can be used as both carrier for PS and photothermal agent for PTT due to its large surface area and intrinsic high NIR absorbance [35e41], is combined with coreeshell structured UCNPs via long-chain joint molecule, the resulting theranostic nanoplatform will exhibit both high fluorescent intensity for imaging and improved therapeutic effect owing to the combination of PDT and PTT. Building from these ideas, herein we design and develop a multifunctional nanocomposite, which is synthesized by covalently grafting coreeshell structured UCNPs with NGO via bifunctional polyethylene glycol (PEG), and then loading phthalocyanine (ZnPc) on the surface of NGO. Benefiting from the unique hybrid nanostructure, this nanocomposite integrates the multifunctions of PDT, PTT and UCL imaging into a single nanoplatform, which could act as an integrated theranostic probe for UCL image-guided combinatorial PDT/PTT of cancer (Scheme 1). Remarkably, both imaging and dual-mode treatments in this nanoplatform are stimulated by light, which exhibits remarkable advantages in terms of enhancing cancer killing specificity and reducing side effects since only the tumor lesion exposed to the light, in comparison to conventional cancer therapies. Moreover, a synergistic effect of combined noninvasive photodynamic and photothermal therapy is expected to improve the therapeutic efficiency, decrease the dosage-limiting toxicity and tissue damage by over-heating. 2. Materials and methods 2.1. Materials Rare-earth oxides RE2O3 (99.99%) (RE ¼ Y, Yb, Er, Tm) were purchased from Science and Technology Parent Company of Changchun Institute of Applied Chemistry. Oleic acid (>90%), 1-octadecene (ODE; >90%), 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC), poly(allylamine) and zinc phthalocyanine were purchased from Aldrich. Amino carboxylic PEG was purchased from Sunbio Inc. All above chemicals were used directly without further purification. Rare earth chloride (RECl3) stock solutions of 0.2 M were obtained by dissolving the corresponding metal oxide in hydrochloric acid under heating with agitation. 2.2. Synthesis of therapeutic probe UCNPs-NGO/ZnPc 2.2.1. Synthesis of coreeshell NaYF4: Yb3þ, Er3þ, Tm3þ/NaYF4 UCNPs Hexagonal-phase NaYF4: Yb3þ, Er3þ, Tm3þ core nanoparticles were synthesized following previously reported method [42e44]. In details, 2 mL solution of RECl3 (0.2 M, RE ¼ Y, Yb, Er, Tm) in methanol was added to a 50 mL flask containing 3 mL oleic acid and 7 mL 1-octadecene. The solution was heated to 110 C for 30 min with vigorous magnetic stirring to remove water and oxygen under argon flow protection, and then heated to 160 C for 30 min. After the solution was cooled to room temperature, a 5 mL methanol solution of NH4F (1.6 mmol) and NaOH (1 mmol) was added, and then the solution was kept at 50 C for 30 min. After methanol was evaporated, the solution was heated to 300 C under an argon atmosphere for 1.5 h and then cooled to room temperature. The nanocrystals were precipitated by addition of ethanol, collected after centrifugation, and washed with ethanol for several times, and dispersed in cyclohexane. The NaYF4 shell precursor was prepared by mixing 2 mL methanol solution of YCl3 (0.2 M), 3 mL oleic acid and 7 mL 1-octadecene, and the solution was heated to 110 C for 30 min, and then to 160 C for 30 min. Next, the solution was cooled to 50 C. The core nanoparticles in 4 mL cyclohexane were added along with 5 mL methanol solution of NH4F (1.6 mmol) and NaOH (1 mmol), and the solution was
stirred at 50 C for 30 min. After the removal of cyclohexane and methanol, the solution was heated to 300 C under argon for 1.5 h and cooled to room temperature. The nanocrystals were collected after centrifugation. 2.2.2. Synthesis of amino-functionalized coreeshell NaYF4: Yb3þ, Er3þ, Tm3þ/NaYF4 UCNPs In order to improve the efficiency of phase transfer, we first synthesized the ligand-free UCNPs following the pervious report [45]. The obtained coreeshell nanoparticles were dispersed in a 10 mL aqueous solution, and stirred for 12 h while maintaining the pH at 4 by adding a solution of HCl (0.1 M). Then the solution was extracted with water diethyl ether three times to remove the oleic acid. The ligandfree UCNPs in the water were obtained by centrifugation after precipitation with acetone. Finally the particles were dispersed in water. Subsequently, 0.1 mL poly(allylamine) 20% solution in water was dispersed in 10 mL water, then the ligand-free UCNPs were added dropwise into poly(allylamine) solution and stirred vigorously over 48 h at room temperature. After centrifugation, UCNPs-NH2 was redispersed in water, and the solution was stable for several days without obvious aggregation. 2.2.3. Synthesis of PEG-functionalized NGO GO was prepared following a modified Hummers method [46,47]. NaOH (0.12 g/ mL) was added to 10 mL GO aqueous suspension (w3 mg/mL), and sonicated for about 4 h. The resulting solution was neutralized, purified by repeated rinsing and centrifugation. The obtained GOeCOOH (1 mg/mL) solution was then mixed with an aqueous solution of amino carboxylic PEG (3 mg/mL). After sonicate for 5 min, EDC was then added to the mixture in two equal portions to give a final concentration of 1 mg/mL totally. The resulting solution was stirred at room temperature for 24 h and then centrifuged filtration through Amicon centrifugal filters (Millipore) with 5 kDa and washed with water to remove the amino carboxylic PEG. 2.2.4. Synthesis of UCNPs-NGO NGO-PEG (0.5 mg/mL) was mixed with a 1 mL aqueous solution of EDC (0.03 mmol), then sonicated for 30 min at room temperature. Subsequently, UCNPsNH2 (0.5 mg/mL) solution was added dropwise into above mixture and stirred vigorously for 24 h. UCNPs-NGO composites were centrifuged and washed with water. 2.2.5. Synthesis of UCNPs-NGO/ZnPc ZnPc (10 mg) was dissolved in 100 mL DMF. After leaving for 24 h, the supernatant was decanted. UCNPs-NGO (5 mg) was dispersed by sonication in 10 mL ZnPc DMF solution. After stirring for 48 h, UCNPs-NGO/ZnPc was obtained by centrifugation, and washed with ethanol to remove excess ZnPc. The resulting UCNPs-NGO/ ZnPc was freeze-dried. 2.3. Detection of singlet oxygen The generation of singlet oxygen was detected by the chemical probe DPBF. In a typical experiment, 20 mL DPBF solution (10 mM) was added to 2 mL UCNPs-NGO/ ZnPc solution, and then transferred into a 10 mm cuvette. The mixture was irradiated with a 630 nm laser at a power density of 50 mW cm2 for 10 min, and the absorption intensity of DPBF at 410 nm was recorded every minute. For comparison, the absorbance of free ZnPc and DPBF mixture was also recorded at the same conditions. 2.4. Cytotoxicity assay The human nasopharyngeal epidermal carcinoma KB cell and human cervix cancer cell line HeLa cell, provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China), were cultured in RPMI-1640 culture medium containing 10% FBS and 1% penicillin/streptomycin at 37 C under 5% CO2. The cells were seeded in 96well plates (5 104 cells per well) for 24 h, and then different concentrations of UCNPs-NGO (0, 20, 40, 80, 160 and 320 mg/mL, diluted in RPMI 1640) were added to the wells. After incubation for 24 h, 10 mL MTT solution (5 mg/mL MTT in PBS, pH 7.4) was added to each well and the plate was incubated for an additional 4 h. After removing the medium, the wells were washed by PBS, and then the intracellular formazan crystals were extracted into 100 mL DMSO. The absorbance was recorded at 490 nm by a plate reader, and the cell viability could be calculated from the average value of six parallel wells. 2.5. PDT and PTT treatments Hela cells were precultured in 96-well plates at 5 104 cells per well for 24 h and then added UCNPs-NGO/ZnPc at different concentrations from 0 to 320 mg/mL. After 24 h of incubation, the cells were washed twice with PBS and irradiated using 808 nm NIR laser at a power density of 2 W cm2 for 10 min. Then, the cells were allowed to incubate for another 24 h. Thereafter, the standard MTT assay was carried out to determine the cell viabilities. The method of detecting the effect of PDT treatment was the same as that used in PTT treatments, except that we used 630 nm laser at a power density of 50 mW cm2 for irradiation.
Y. Wang et al. / Biomaterials 34 (2013) 7715e7724 Scheme 1. (a) Schematic illustration of the synthetic procedure for UCNPs-NGO: Numbers of coreeshell structured UCNPs being covalently grafted with NGO via bifunctional polyethylene glycol; (b) Schematic illustration of UCNPsNGO/ZnPc as a multifunctional theranostic nanoplatform for cancer treatment. 7717
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Y. Wang et al. / Biomaterials 34 (2013) 7715e7724 2.8. AO/EB staining of HeLa cells After cultured with 80 mg/mL UCNPs-NGO/ZnPc nanocomposites for 24 h, the cells (control and experimental groups) were treated without or with single or combinational laser. After another 24 h incubation, 10 mL of 50 mg/mL AO and EB mixed dye solution in PBS was added to each Petri dish and co-cultured with cells in darkness for 20 min at room temperature. Thereafter, the cells were washed with PBS to remove excess dye and detached cells. Fluorescence microscope was used to monitor the cell morphology. AO emissions were measured in the green channel when excited by 488 nm, and EB emissions were collected in the red channel when excited by 543 nm.
3. Results and discussion 3.1. Preparation and characterization
Fig. 1. TEM image of monodisperse UCNPs-NH2 nanoparticles in water.
For synergistically enhanced anti-cancer effect of the combination of PDT and PTT, Hela cells were seeded in 96-well plates at 5 104 cells per well for 24 h, and then 40 or 80 mg/mL UCNPs-NGO/ZnPc nanocomposites were added. After 24 h of incubation, the cells were exposed to the 808 nm NIR laser at a power density of 2 W cm2 for 10 min. Subsequently, cells were irradiated by 630 nm laser at a power density of 50 mW cm2 for 10 min. After another 24 h of incubation, cell viability was measured according to the standard MTT assay method. 2.6. UCL imaging of living cells For confocal imaging, KB cells and HeLa cells were plated on 14 mm glass coverslips under 100% humidity and allowed to adhere for 24 h. The cells were stained with 200 mg/mL UCNPs-NGO at 37 C for 2 h. Prior to imaging, the coverslips were washed twice with PBS in order to remove excess UCNPs-NGO. Confocal imaging of cells was performed with a modified Olympus FV1000 laser scanning upconversion luminescence microscope (LSUCLM) equipped with a continuous-wave (CW) laser at 980 nm (Connet Fiber Optics, China). Upconversion luminescence signals were detected in the green channel (520e560 nm) and red channel (600e700 nm). 2.7. UCL imaging in vivo of mice All animal studies were performed in accordance with institutional and national guidelines. Kunming mouse (18 g) was anesthetized (with 10% chloral hydrate 100 L) and was subcutaneously injected with the UCNPs-NGO. Subsequently, the mouse was imaged by a modified in vivo Maestro whole-body imaging system with an external 980 nm laser as the excitation source.
For highly efficient UCL, coreeshell structured Tm3þ/Er3þ/Yb3þ co-doped NaYF4@NaYF4 nanocrystals were synthesized as fluorescent probe using previously reported method [42e44]. The average diameters of the core and coreeshell UCNPs were determined to be 28 nm and 40 nm by transmission electron microscopy (TEM), respectively, as presented in Fig. S1 (Supporting Information). The X-ray diffraction (XRD) pattern showed that the nanoparticles were hexagonal b-NaYF4 (Fig. S2). The UCNPs were further converted into hydrophilic UCNPs-NH2 via a ligand exchanging process using poly(allylamine) (PAAm) as surface coating agent to replace the original oleic acid ligands. The TEM image (Fig. 1) demonstrated high monodispersity of UCNPs-NH2 in water, which was ideal for biological applications. In order to endow the obtained UCNPs with multimodal therapeutic functions, NGO as both PTT agent and carriers for PS was covalently grafted with UCNPs via a carbodiimide cross-linking reaction between the amino group of UCNPs-NH2 and the carboxyl group of NGO-PEG following the protocol as illustrated in Scheme 1a. NGO-PEG was obtained by covalently grafting amino carboxylic PEG onto NGO sheet, and the left carboxylic groups at the PEG terminals were available for fluorescent probes. The successful PEG functionalization was evidenced by Fourier-transform infrared spectroscopy (FT-IR; Fig. S3). The morphology of NGOPEG was characterized by atomic force microscope (AFM, Fig. 2a). NGO-PEG was almost single layered sheet with average topographic height of w1.2 nm, as shown in Fig. 2b. To verify the potential of using NGO in PTT, we detected the temperature variation of NGO-PEG solution exposed to an 808 nm NIR laser at a power density of 2 W/cm2 using a thermal imaging camera (Fig. 3). Before NIR exposure, the maximum temperature was about 23 C, but the temperature of NGO-PEG solution increased rapidly to 58 C within 3 min after exposing to NIR light (Fig. S4), which indicated that the NGO could rapidly and efficiently convert the 808 nm laser energy
Fig. 2. (a) AFM image of NGO-PEG deposited on mica substrate, and (b) the height profile of the AFM image, showing NGO-PEG was almost single layered sheet.
Y. Wang et al. / Biomaterials 34 (2013) 7715e7724 Fig. 3. Temperature change images of the NGO-PEG solution exposed to the 808 nm laser for different time periods at a power density of 2 W/cm2, showing NGO could rapidly and efficiently convert the 808 nm laser energy into thermal energy.
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Fig. 4. (a) Upconversion emission spectra of UCNPs-NH2 and UCNPs-NGO excited with a CW 980 nm laser. (b) Photographs of UCNPs-NGO under ambient light (left) and under CW 980 nm laser irradiation (right).
Fig. 5. Time-course generation of singlet oxygen by UCNPs-NGO/ZnPc and free ZnPc detecting by the bleaching of DPBF absorption at 410 nm under 630 nm laser irradiation.
into thermal energy. The reason for selecting PEG as linker was twofold. On one hand, PEG has good compatibility with biological system, which renders NGO high aqueous solubility and stability in physiological solutions including serum [48]. On the other hand, bonding UCNPs at the end of the long chain of PEG molecules can reduce the fluorescence quenching by NGO. The emission spectra of UCNPs-NH2 and UCNPs-NGO (Fig. 4a) displayed characteristic green, red (in web version) and NIR emission bands under CW excitation at 980 nm. The visible emission bands could be assigned to the 2H11/2 / 4I15/2, 4S3/2 / 4I15/2 and 4F9/2 / 4I15/2 transition of Er3þ ions, respectively. The NIR emission band around 808 nm was attributed to Tm3þ transitions from 3H4 to 3H6, which was more suitable for in vivo imaging owing to the higher penetration depth. Although the emission intensity of UCNPs-NGO was a little weaker than that of UCNPs-NH2 owing to the part absorption by NGO, the retained emission was still strong enough to be observed by naked eye (Fig. 4b). This result demonstrates that our strategy is highly effective to avoid quenching UCL by therapeutic agent, endowing the nanoplatform with both high fluorescent intensity and high therapeutic effect. To achieve multimodal efficient therapy, aromatic ZnPc with high optical absorption coefficient in the phototherapeutic window of 600e800 nm was used as a model PS, and loaded on the surface of UCNPs-NGO for PDT by mixing the ZnPc/DMF solution with the UCNPs-NGO aqueous solution for 48 h. The UVevis spectra of UCNPs-NGO/ZnPc showed the absorption bands were red shifted and broadened compared with those of ZnPc dissolved in DMF (Fig. S5). Such result manifested a strong interaction of ZnPc with UCNPs-NGO via pep stacking. Following the method reported by
Shi’s group [49], the loading efficiency was calculated to be 11%. Such high PS loading efficiency indicated that UCNPs-NGO was a promising PS nanocarrier for PDT. Singlet oxygen generation was the critical step in PDT, so the 1O2 generation ability of UCNPs-NGO/ ZnPc was a very important index to illustrate its application potential in PDT. We monitored the singlet oxygen production of UCNPs-NGO/ZnPc and free ZnPc by the chemical probe 1,3-
Fig. 6. Cell viability of HeLa and KB cells incubated with UCNPs-NGO at different concentration for 24 h, respectively, showing the low cytotoxicity of UCNPs-NGO.
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Fig. 7. Upconversion luminescence imaging at green channel (lem ¼ 520e560 nm) and red channel (lem ¼ 600e700 nm) of HeLa and KB cells stained with 200 mg/mL UCNPs-NGO for 2 h at 37 C. Overlay of luminescence image and bright field image were also shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
diphenylisobenzofuran (DPBF), whose absorbance at 410 nm would be diminished in the presence of 1O2. Fig. 5 showed the absorbance of solutions of DPBF and UCNPs-NGO/ZnPc or free ZnPc as a function of exposure time to a 630 nm laser irradiation. The absorbance intensity of DPBF displayed a continuous decrease with 10 min for both UCNPs-NGO/ZnPc and free ZnPc. Although the 1O2 production ability by UCNPs-NGO/ZnPc was weaker than that of free ZnPc owing to the quenching effect by NGO, it still retained about 60% of free ZnPc, which allowed us to use UCNPs-NGO/ZnPc for PDT treatment of cancer cells. 3.2. In vitro cytotoxicity Prior to the theranostic application of UCNPs-NGO nanoplatform, the in vitro cytotoxicity of UCNPs-NGO was assessed using a traditional MTT assay. Fig. 6 showed the in vitro cell viability of human cervix cancer HeLa cell and human nasopharyngeal epidermal cancer KB cell incubated with UCNPs-NGO with different concentration ranging from 20 to 320 mg/mL for 24 h. The cell viabilities were higher than 90% for both HeLa and KB cell, even at the high concentration of 320 mg/mL. These data show satisfactory results for in vitro non-cytotoxicity for all dosages of UCNPs-NGO. Good biocompatibility and low cytotoxicity imply that UCNPsNGO can serve as a theranostic probe for simultaneous UCL imaging and combinatorial PDT/PTT of cancer.
3.3. Upconversion luminescence (UCL) imaging in vitro and in vivo Cellular uptake of UCNPs-NGO was investigated by laser scanning upconversion luminescence microscopy (LSUCLM). After incubation with 200 mg/mL UCNPs-NGO for 2 h at 37 C, an intense UCL signal from 520 to 560 nm and 600e700 nm was observed from the HeLa cells (Fig. 7) under CW excitation at 980 nm. The nanocomposites were mainly located in cytoplasm and perinuclear regions, which indicated that UCNPs-NGO penetrated the cell membrane of living HeLa cells. To further verify whether UCNPsNGO can be used for other cancer cells, KB cells were chosen to uptake the nanocomposites. From the confocal luminescence imaging of KB cells, we can see that UCNPs-NGO has crossed the cell membrane and entered the cytoplasm, which is similar to that of HeLa cells. The above results demonstrated that UCNPs-NGO is promising candidate for cancer cellular labeling and imaging. Encouraged by the results of UCL imaging of cancer cells in vitro, we subcutaneously injected UCNPs-NGO onto the back of a white Kunming mouse, which was then imaged by a modified in vivo Maestro whole-body imaging system with an external 980 nm laser as the excitation source. After injection, a strong UCL signal arose from the place where the nanocomposites were injected (Fig. 8), which revealed that the UCL signal could easily penetrate these tissues of the mouse. More importantly, almost no autofluorescence was detected elsewhere. The result confirms the
Fig. 8. In vivo upconversion luminescence imaging of a white Kunming mouse after subcutaneous injection 200 mg/mL UCNPs-NGO.
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Fig. 9. (a) In vitro photodynamic and photothermal treatment of cancer cell: viability of HeLa cells treated with UCNPs-NGO/ZnPc of different concentrations without and with irradiation by 808 nm or 630 nm laser. (b) In vitro combined photodynamic and photothermal treatment of cancer cell: viability of HeLa cells treated with UCNPs-NGO/ZnPc at 40 mg/mL and 80 mg/mL. Black, red, blue, and green bars represent cells without any light irradiation, with only 808 nm laser irradiation, with only 630 nm laser irradiation, and with both 808 nm and 630 nm laser irradiation, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
capability of UCNPs-NGO nanocomposites for high contrast UCL imaging in vivo. 3.4. Photodynamic therapy and photothermal therapy Cell destruction by both PDT and PTT was studied using HeLa cells. Cells were incubated with UCNPs-NGO/ZnPc at a series of concentrations for 24 h and then irradiated with either 630 nm or 808 nm laser for 10 min. In a concentration-dependent manner, without light exposure, UCNPs-NGO/ZnPc exhibited negligible dark toxicity to HeLa cells at concentrations up to 320 mg/mL (Fig. 9a). As indicated by the 1O2 generation ability studies, singlet oxygen was produced by irradiating of UCNPs-NGO/ZnPc with 630 nm laser at a density of 50 mW/cm2. Therefore, when HeLa cells were exposed to laser, the cell viability decreased significantly with the increase of the concentration of UCNPs-NGO/ZnPc, manifesting the feasibility of the nanocomposites to destroy the cancer cells by 1O2. As for the effect of PTT, the cell viability for UCNPs-NGO/ZnPc at the concentration from 20 to 320 mg/mL decreased from about 95% to 30% after irradiating for 10 min with 808 nm laser, demonstrating an obvious photothermal efficacy. These results clearly demonstrate that UCNPs-NGO/ZnPc has a high possibility for PDT and PTT. A cell viability assay was conducted at two UCNPs-NGO/ZnPc concentrations (40 and 80 mg/mL, denoted as group A and B) (Fig. 9b) in order to further quantify the synergistically enhanced anti-cancer effect of the combination of the two treatment approaches. The control cells, which were incubated with different concentration of UCNPs-NGO/ZnPc without any light irradiation, showed nearly no damage. After irradiation of 630 nm laser, the cell viability of group A and B decreased to about 75% and 50%, respectively. Because the power and optical dose of the 630 nm light applied in this experiment were extremely low without causing any noticeable heating of UCNPs-NGO/ZnPc, cell mortality only induced by singlet oxygen generation upon excitation of the photosensitizer. In the same time, NIR laser irradiation decreased the cell viability to about 85% and 75% for the two groups based on the photothermal killing of cancer cells. As expected, when the two treatments were combined, the cell viability was remarkably reduced to 35% and 15%, respectively, which is evidently much lower than that by the individual ones. The remarkably improved therapeutic effect may be attributed to the photothermal effect of graphene that not only can “cook” the cancer cells but also enhance the delivery of PDT agents for improved photodynamic cancer cell
killing [50]. Therefore, UCNPs-NGO/ZnPc can destroy cancer cells more efficiently when used for combined PDT and PTT. Taken together, the nanocomposites as-assembled for an adaptive tumor theranostic platform could be proposed as follows. Preliminarily, UCNPs-NGO/ZnPc would be localized in tumor tissues owing to the enhanced permeability and retention effect and further internalized by tumor cell through the energy-dependent endocytosis process, followed by the formation of endosomes. Meanwhile, the tumor cells could be clearly observed in the UCL image with a high signal-to-background ratio. Then, selectively destroying the tumor cells guided by UCL imaging was carried out in a noninvasive manner using laser irradiation, which minimized damage of the surrounding normal cells. Finally, synergistically enhanced anticancer efficacy of combinatorial PDT/PTT was achieved. Furthermore, for the sake of visualizing the tumorigenic cell ablation efficacy and demonstrating the relevant mechanism in vitro, HeLa cells were incubated with UCNPs-NGO/ZnPc (80 mg/ mL) for 12 h. After a series of treatment with single or combinational laser, the samples were stained with a mixture of the nucleic acid-specific dyes acridine orange (AO) and ethidium bromide (EB) following the standard staining protocols and immediately observed by fluorescence microscopy. It is known that AO can enter live cells and give the nuclei a green fluorescence, while EB is excluded by intact tumor cell membranes but stains the nuclei of dead cells fluorescent orange. So we could assess the cell viability by observing fluorescent microscopic image. In the control experiment, cell viability exhibits no obvious decline, indicating the nanocomposite itself could not lead to cell death without irradiation (Fig. 10aeb). However, it is observed that several cells show a bright orange red color (in web version) after irradiation with 808 nm laser (Fig. 10ced). The damage of cells may be caused by localized hyperthermia. After irradiation of 630 nm laser, the cell viability distinctively decreases, and nearly 40% cell nucleus displays orange red light (in web version) (Fig. 10eef), showing late apoptotic or necrotic character. Such result demonstrates that UCNPs-NGO/ZnPc nanocomposite could kill cells through both photodynamic and photothermal effects under laser irradiation. After combinational treatment with the two lasers, as presented in Fig. 10geh, the necrotic areas appear, and none of the rest cells is alive. This further certified that the combined PDT and PTT multimodal nanoplatform based on UCNPs-NGO/ZnPc nanocomposites was a more efficient therapeutic regimen against cancer cells than either PDT or PTT alone.
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Fig. 10. Representative fluorescent microscopic images of HeLa cells treated with 80 mg/mL UCNPs-NGO/ZnPc with or without single or combinational laser illumination, and then the cells were stained by nucleic acid-specific dyes AO and EB. (aeb) without irradiation; (ced) under irradiation of 808 nm laser; (eef) under irradiation of 630 nm laser; (geh) under combinational laser irradiation.
4. Conclusions We have prepared UCNPs-NGO/ZnPc as a theranostic platform for UCL image-guided combinatorial PDT/PTT of cancer. Cytotoxicity assays demonstrated good biocompatibility and low toxicity of the UCNPs-NGO/ZnPc. The nanocomposite could be used as UCL imaging probe of cells and whole-body animals with high contrast for diagnosis. At the same time, significantly greater cell killing was obtained because UCNPs-NGO/ZnPc was not only excellent nanocarrier to load ZnPc for PDT, also could kill cells by a photothermal effect. Compared with photodynamic or photothermal treatment
alone, the combined treatment showed a synergistic effect, resulting in higher therapeutic efficacy for in vitro cancer therapy. These results highlight that integration of these functionalities endows UCNPs-NGO/ZnPc with the potential promise for cancer theranostics. Acknowledgments The authors are grateful to the financial aid from the National Natural Science Foundation of China (Grant Nos. 21001101, 21071140, 91122030 and 21210001), ‘863’-National High
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Technology Research and Development Program of China (Grant No. 2011AA03A407) and National Natural Science Foundation for Creative Research Group (Grant No. 21221061). We wish to thank Prof. F. Y. Li (FU) for kindly providing UCL imaging measurements. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2013.06.045. References [1] Greco F, Vicent M. Combination therapy: opportunities and challenges for polymer-drug conjugates as anticancer nanomedicines. Adv Drug Deliv Rev 2009;61:1203e13. [2] Lane D. Designer combination therapy for cancer. Nat Biotechnol 2006;24: 163e4. [3] Wu S, Han G, Milliron DJ, Aloni S, Altoe V, Talapin DV, et al. Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals. Proc Natl Acad Sci U S A 2009;106:10917e21. [4] Park YI, Kim JH, Lee KT, Jeon KS, BinNa H, Yu JH, et al. Nonblinking and nonbleaching upconverting nanoparticles as an optical imaging nanoprobe and T1 magnetic resonance imaging contrast agent. Adv Mater 2009;21: 4467e71. [5] Li Z, Zhang Y, Jiang S. Multicolor core/shell-structured upconversion fluorescent nanoparticles. Adv Mater 2008;20:4765e9. [6] Liu Q, Sun Y, Yang T, Feng W, Li C, Li F. Sub-10 nm hexagonal lanthanidedoped NaLuF4 upconversion nanocrystals for sensitive bioimaging in vivo. J Am Chem Soc 2011;133:17122e5. [7] Auzel F. Upconversion and anti-stokes processes with f and d ions in solids. Chem Rev 2004;104:139e74. [8] Wang F, Liu XG. Recent advances in the chemistry of lanthanide-doped upconversion nanocrystals. Chem Soc Rev 2009;38:976e89. [9] Wang F, Banerjee D, Liu YS, Chen XY, Liu XG. Upconversion nanoparticles in biological labeling, imaging, and therapy. Analyst 2010;135:1839e54. [10] Haase M, Schäfer H. Upconverting nanoparticles. Angew Chem Int Ed 2011;50:5808e29. [11] Zhou J, Liu Z, Li FY. Upconversion nanophosphors for small-animal imaging. Chem Soc Rev 2012;41:1323e49. [12] Nyk M, Kumar R, Ohulchanskyy TY, Bergey EJ, Prasad PN. High contrast in vitro and in vivo photoluminescence bioimaging using near infrared to near infrared upconversion in Tm3þ and Yb3þ doped fluoride nanophosphors. Nano Lett 2008;8:3834e8. [13] Jalil RA, Zhang Y. Biocompatibility of silica coated NaYF(4) upconversion fluorescent nanocrystals. Biomaterials 2008;29:4122e8. [14] Hu H, Yu M, Li F, Chen Z, Gao X, Xiong L, et al. Facile epoxidation strategy for producing amphiphilic up-converting rare-earth nanophosphors as biological labels. Chem Mater 2008;20:7003e9. [15] Idris NM, Li ZQ, Ye L, Sim EKW, Mahendran R, Ho PCL, et al. Tracking transplanted cells in live animal using upconversion fluorescent nanoparticles. Biomaterials 2009;30:5104e13. [16] Xiong LQ, Chen ZG, Yu MX, Li FY, Liu C, Huang CH. Synthesis, characterization, and in vivo targeted imaging of amine-functionalized rare-earth upconverting nanophosphors. Biomaterials 2009;30:5592e600. [17] Xiong LQ, Chen ZG, Tian QW, Cao TY, Xu CJ, Li FY. High contrast upconversion luminescence targeted imaging in vivo using peptide-labeled nanophosphors. Anal Chem 2009;81:8687e94. [18] Yu M, Li F, Chen Z, Hu H, Zhan C, Yang H, et al. Laser scanning up-conversion luminescence microscopy for imaging cells labeled with rare-earth nanophosphors. Anal Chem 2009;81:930e5. [19] Boyer JC, Manseau MP, Murray JI, van Veggel CJM. Surface modification of upconverting NaYF4 nanoparticles with PEG-phosphate ligands for NIR (800 nm) biolabeling within the biological window. Langmuir 2010;26: 1157e64. [20] Kobayashi H, Kosaka N, Ogawa M, Morgan NY, Smith PD, Murray CB, et al. In vivo multiple color lymphatic imaging using upconverting nanocrystals. J Mater Chem 2009;19:6481e4. [21] Wang M, Mi CC, Wang WX, Liu CH, Wu YF, Xu ZR, et al. Immunolabeling and NIR-excited fluorescent imaging of HeLa cells by using NaYF4:Yb, Er upconversion nanoparticles. ACS Nano 2009;3:1580e6. [22] Yu X, Li M, Xie M, Chen L, Li Y, Wang Q. Dopant-controlled synthesis of watersoluble hexagonal NaYF4 nanorods with efficient upconversion fluorescence for multicolor bioimaging. Nano Res 2010;3:51e60.
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