FULL PAPER DOI: 10.1002/asia.201301695
One-Pot Template-Free Synthesis of NaYF4 Upconversion Hollow Nanospheres for Bioimaging and Drug Delivery Gan Tian,[a, c] Longsheng Duan,[a] Xiao Zhang,[a, b] Wenyan Yin,*[a] Liang Yan,[a] Liangjun Zhou,[a, d] Xiaodong Liu,[a] Xiaopeng Zheng,[a, d] Jinxia Li,[a] Zhanjun Gu,*[a] and Yuliang Zhao*[a, b, c] Abstract: Hollow-structured nanomaterials with fluorescent properties are extremely attractive for image-guided cancer therapy. In this paper, sub100 nm and hydrophilic NaYF4 upconversion (UC) hollow nanospheres (HNSs) with multicolor UC luminescence and drug-delivery properties were successfully prepared by a facile one-pot template-free hydrothermal route using polyetherimide (PEI) polymer as the stabilizing agent. XRD,
SEM, TEM, and N2-adsorption/desorption were used to characterize the asobtained products. The growth mechanism of the HNSs has been systematically investigated on the basis of the Ostwald ripening. Under 980 nm excitation, UC emissions of HNSs can be Keywords: drug delivery · hollow nanospheres · luminescence · onepot synthesis · Ostwald ripening
Introduction
used as luminescent probes for biological imaging or as tracers for drug-release monitoring.[1c] Lanthanide (Ln)-doped upconversion (UC) luminescent nanomaterials, which can convert low-energy (near-infrared (NIR) photon) excitation to higher-energy (UV-visible light) emission by means of multiple absorption or energy transfer,[2] have been increasingly proposed as next-generation bioprobes for bioimaging since they have many advantages over conventional organic dye markers and quantum dots, such as high physicochemical stability, low toxicity, reduced photodamage to living organisms, high signal-to-noise ratio, and strong tissue penetration ability.[2b] Therefore, the design and fabrication of hollow UC nanostructures would undoubtedly be of great importance in the field of image-guided drug delivery.[2d] Generally, a template-assisted strategy that involves the coating of templates with nanocrystals (precursors or targeted products) and then removal of the templates has been widely employed for the preparation of hollow-structured UC nanomaterials since the templating method offers important advantages, including easy operation and well-defined structural features of the products with narrow size distribution.[3] Nonetheless, the disadvantages of this method, such as tedious synthetic procedures and low yields, have largely impeded the upscaled production and largescale applications. Moreover, collapse of some fraction of the hollow structures always occurs during the templatecore removal process through high-temperature calcination (e.g., carbon spheres) or chemical dissolution (e.g., using HF to etch silica spheres). Unfortunately, the products obtained from the high-temperature calcination processes com-
Recently, luminescent hollow-structured materials have attracted considerable attention owing to their unique properties and potential applications in biomedical fields.[1] Relative to conventional biomedical systems, these novel multifunctional materials not only provide a cavity structure for the controlled storage/delivery of therapeutic drugs but also possess luminescence properties, which means they can be
[a] Dr. G. Tian,+ Dr. L. Duan,+ Dr. X. Zhang, Dr. W. Yin, Dr. L. Yan, Dr. L. Zhou, Dr. X. Liu, Dr. X. Zheng, Dr. J. Li, Dr. Z. Gu, Prof. Y. Zhao CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics Chinese Academy of Sciences, Beijing 100049 (P.R. China) E-mail: [email protected] [email protected] [email protected] [b] Dr. X. Zhang, Prof. Y. Zhao National Center for Nanosciences and Technology of China Beijing, 100190 (P.R. China) [c] Dr. G. Tian,+ Prof. Y. Zhao College of Chemistry, Sichuan University Chengdu, 610064 (P.R. China) [d] Dr. L. Zhou, Dr. X. Zheng College of Materials Science and Opto-electronic Technology Graduate University of Chinese Academy of Sciences Beijing 100049 (P.R. China) [+] These authors contributed equally. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201301695.
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tuned by a simple change of the concentration or combination of various upconverters. As a result, the PEIcoated HNSs could be used as efficient probes for in vitro upconversion luminescence (UCL) cell imaging. Furthermore, a doxorubicin storage/release behavior and cancer-cell-killing ability investigation reveal that the product has the potential to be a drug carrier for cancer therapy.
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pletely lose the chemical functional groups located on their water solubility and good biocompatibility show intense surfaces, thereby resulting in poor water solubility and thus multicolor upconversion luminescence (UCL) under 980 nm depriving their prospects in the biological field.[3b] AlternaNIR laser excitation. Considering the presence of the cavity tively, to simplify the preparation procedure, a template-free in the hollow nanostructures, the material can be used as an process has also been developed for the construction of effective drug carrier when using doxorubicin (DOX) as hollow UC structures.[4] Zhao et al. reported a facile solua model drug. The drug-loading and release properties, cytotion-phase synthesis of NaYF4 :Yb/Er(Tm) UC hollow toxicity, and therapeutic effects are investigated in detail. spheres based on the nanoscale growth induced by the Kirkendall effect.[4a] Lin et al. employed the same strategy for controllable synthesis of NaYF4 :Yb/Er hollow spheres[4b] Results and Discussion and core–shell-structured Yb(OH)CO3@YbPO4 :Er hollow Morphology and Structure microspheres.[4c] However, all these synthetic schemes require the use of their corresponding oxides or carbonates as Large-scaled NaYF4 nanospheres can be achieved by means the precursor. Therefore, it is still significant and urgent to of hydrothermal treatment at 200 8C for 12 h using PEI as explore other template-free strategies that aim toward the surfactant. As can be seen in Figure 1a, these nanoa facile one-step synthetic approach for obtaining hollow spheres with a size of (50 6) nm (Figure S1, Supporting Instructures. Currently, utilization of the well-known classical formation) have a rugged and bumpy surface (Figure S2, physical phenomenon known as the Ostwald ripening mechSupporting Information). The broken nanospheres are hemianism provides an optional opportunity for one-pot synthespherical or bowl-like in shape, which suggests the presence sis of Ln-based luminescent hollow structures.[5] This desiraof a characteristic cavity in hollow structures; this could also be confirmed by the TEM image presented in Figure 1a, ble strategy simplifies the synthetic procedure and improves inset. The N2 adsorption–desorption isotherm in Figure 1b the production yield.[5a] So far, one-pot fabrication of hollow UC structures that undergo the Ostwald ripening mechashows that the isotherm is type II and reveals the existence nism has been lingering far behind the significant progress of mesoporosity in the sample owing to the presence of hysthat has been made in the template-synthesis method. In adteresis in the adsorption.[5c] The Brunauer–Emmett–Teller dition, the as-prepared products are usually microscaled, (BET) surface area is about 20.02 m2 g1, and the pore [5c] which makes them unsuitable for biological applications. volume is 0.126 cm3 g1 as calculated from N2 isotherms at As a result, developing a facile strategy based on the Ost77 K. The pore-size distribution (Figure 1b, inset) was calcuwald ripening mechanism to fabricate sub-100 nm and highly water-soluble UC hollow nanospheres (HNSs) is still a big challenge. In this work, we report an efficient, one-pot route to synthesize sub-100 nm water-soluble NaYF4-based UC HNSs under hydrothermal conditions based on a gas-bubble-assisted Ostwald ripening mechanism from solid spheres to hollow spheres.[6] In this reaction system, we used ethylene glycol (EG) as the main solvent and polyethylenimine (PEI) as the structure-stabilizing agent. The solvent EG with abundant hydroxyl groups could also be seen as the second stabilizing agent to assist PEI for efficiently controlling the nucleation and growth of the NaYF4 crystal, thereby largely decreasing the size of the self-assembled Figure 1. Materials characterization: a) SEM image, inset: a typical TEM image showing the hollow structures; solid spheres and thus achieving b) N adsorption–desorption isotherm, inset: pore-size distribution curve; c) XRD patterns; and d) FTIR spec2 nanoscaled HNSs. The PEI- trum of the as-obtained NaYF4 hollow nanospheres, inset: photograph of the as-prepared PEI-coated NaYF4 coated UC HNSs with high HNSs dispersed in deionized (DI) water (1 mg mL1).
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lated using the Barrett–Joyner–Halenda (BJH) distribution from the adsorption branch, thus indicating the pore-size distributions in the mesoporous (2–50 nm) and macroporous (> 50 nm) regions with a maximum peak pore diameter of approximately 35 nm. The XRD pattern in Figure 1c indicates a high crystallinity of the as-prepared nanoparticles. The cubic NaYF4 phase (JCPDS no. 77-2042) is the dominant phase, but a small quantity of hexagonal NaYF4 phase (JCPDS no. 16-0334) is also observed. Previous reports have demonstrated the stabilizing agent PEI could strongly coordinate with Y3 + ions, which would decrease both the nucleation process and nuclei growth and thus suppress the formation of hexagonal NaYF4 phase.[7] Nevertheless, the prolonged heat treatment benefits the phase transformation from the metastable cubic phase to the more stable hexagonal phase.[8] The presence of PEI polymer on the surface of the HNSs was confirmed by FTIR spectroscopy as shown in Figure 1d. The bands at 2929 and 2852 cm1 are attributed to the stretching vibrations of the CH2 group, respectively. In addition, peaks centered at 1636 and 1431 cm1 are internal vibrations of the amide group, and the peak at 1533 cm1 is the characteristic asymmetric vibration of NH2 group,[4b, 7a] thus demonstrating the existence of the PEI polymer. Owing to the presence of hydrophilic PEI on the surface, these nanospheres could be readily dispersed in deionized water, thus forming a nearly transparent aqueous solution (Figure 1d, inset).
needed for structure evolution from a solid to hollow structure by means of an Ostwald ripening process is up to at least 24 h.[9] In the present work, HNSs can be obtained within 12 h. The formation process can be explained by the possible reaction processes proposed by Equations (1–4) in this system, whereby the gas bubbles generated under high temperature and pressure would play an important role in the formation of HNSs.[6c] YNO3 þ PEI ! Y3þ @PEI complex þ 3 NO3 4 NH4 F þ NaCl þ 3 NO3 þ Y3þ @PEI ! NaYF4 @PEI þNH4 Cl þ 3 NH4 NO3 D
NH4 Cl ! NH3 " þHCl " D
NH4 NO3 ! N2 O " þ2 H2 O
ð2Þ ð3Þ ð4Þ
In this reaction system, the surfactant PEI has good affinity towards Y3 + and will form a Y3 + @PEI complex at the very beginning [Eq. (1)].[7a] An appropriate PEI amount is indispensable for the formation of NaYF4 HNSs.[5c] In a series of control groups, we find that PEI-free products are bulk polyhedron and predominantly irregular nanoparticles. Also, amounts of PEI that exceed 0.20 g do not benefit the formation of HNSs but produce solid spheres with smooth surfaces (Figure S3, Supporting Information). Owing to the low free Y3 + concentration in the solution that arises from the weak dissociation tendency of the Y-PEI complex,[4b] the slow nucleation of NaYF4 [Eq. (2)] is the rate-determining step. Simultaneously, a large amount of gas bubbles could be generated in the reaction system [Eqs. (3) and (4)] and serve as nucleation centers for the freshly formed nuclei.[6b] These smaller NaYF4 nuclei would assemble and form the solid NaYF4 spheres to minimize the interfacial energy.[5c, 9b] These kinds of metastable solid spheres can be obtained in large quantities by adjusting the reaction time to 2 h (Figure 2b). Relative to previously reported work,[5, 6] the self-assembled solid spheres here are much smaller. In our procedure, we used an EG/H2O system as the reaction media, the addition of a small amount of H2O is crucial for the oriented aggregation of NaYF4 nuclei (Figure S4, Supporting Information) and benefits the Ostwald ripening process.[10] EG with abundant hydroxyl groups could also coordinate with Y3 + ions and thus influence the NaYF4 nucleation and selfassembly process, thereby causing the formation of solid spheres with nanoscale size. Products prepared in parallel without EG are predominantly bulk prisms (Figure S5, Supporting Information). As the hydrothermal process progresses, the system energy is high enough to trigger the Ostwald ripening process.[6c, 9a] During this period, smaller-sized, less crystalline, and higher-surface-energy nuclei in the inner cores will be gradually dissolved and merged with nanoparticles in the outer surface, thereby producing channels that connect the inner and outer spaces in the NaYF4 shells (Figure 2c). Finally, after further prolonging the reaction to 12 h, the cores within the hollow spheres were consumed com-
The Proposed Formation Mechanism The possible formation process of NaYF4 UC HNSs can be explained by the Ostwald ripening process; the whole process is illustrated in Figure 2a. Usually, the reaction time
Figure 2. Proposed formation mechanism: a) schematic illustration of the proposed mechanism for the formation of NaYF4 HNSs by means of Ostwald ripening. TEM images of NaYF4 products at different growth times: b) 2, c) 6, and d) 12 h, respectively. e) Time-dependent XRD patterns of the products.
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tributed to 4H11/2 !4I15/2 and 4S3/2 !4I15/2 transitions of Er3 + ions, respectively, whereas the red band that peaked at 653 nm is ascribed to an 4F9/2 !4I15/2 transition.[2a] By varying the Yb3 + doping level, the color output from green to yellow could be realized. Increasing the amount of Yb3 + dopants decreased the Yb···Er interatomic distance and thus facilitated back-energy transfer from Er3 + to Yb3 + , thereby decreasing the transition possibility of green (4H11/2, 4S3/2 ! 4 I15/2) light emissions and increasing the ratio of red to green emission.[3b, 7b] For Yb/Tm co-doped HNSs, six characteristic bands centered at 361, 450, 475, 650, 725, and 800 nm belong to the 1D2 !3H6, 1D2 !3F4, 1G4 !3H6, 1G4 !3F4, 3F3 ! 3 H6, and 3H4 !3H6 transitions of Tm3 + ions, respectively,[2b] and the sample exhibits a blue emission (Figure 3b, (4)). In the dual-emitter dopant system, characteristic peaks that belong to each emitter can be clearly observed (Figures 3a, (4) and (5)) and a nearly white-light emission (Figures 3b, (4) and (5)) is achieved in the dual-emission process.
pletely and hollow nanospheres formed (Figure 2d). The XRD patterns in Figure 2e show that thermodynamically more stable hexagonal-phase NaYF4 appeared, and the relative intensity increased in a time-dependent manner, partly supporting the dissolution–recrystallization involved in the Ostwald ripening process, whereby the thermodynamically more stable structure would be preferentially generated during the recrystallization process.[5c] UCL Properties NaYF4 is considered one of the most efficient host matrixes for UC nanomaterials owing to its low phonon frequency, which can minimize nonradiative losses and maximize the radiative emission intensity.[2d, 8c] The UCL of upconverterdoped samples can be clearly observed under the excitation of a 980 nm continual-wave laser, and the color output could be manipulated by adjusting the combination or concentration of the doped upconverters. The UCL spectra and their corresponding photographs of Yb/Er, Yb/Tm, or Yb/ Er,Tm co-doped NaYF4 HNSs are given in Figure 3. For the Yb/Er co-doped HNSs, the weak blue emission band that peaked at 410 nm can be assigned to an 2H9/2 !4I15/2 transition, and the green bands centered at 521 and 541 nm are at-
Cell Viability and UCL Cell Imaging Cytotoxicity is an essential concern when it comes to the development of nanomaterials for biomedical imaging and cancer-therapy applications.[11] The viability of KB cells after exposure to NaYF4 :Yb/Er UC HNSs was measured by a standard Cell Counting Kit-8 (CCK-8) assay. Figure 4a
Figure 4. Cytotoxicity test and UCL cell imaging: a) Cell viabilities of KB cells incubated with NaYF4 :Yb/Er UC HNSs at different concentrations for 24 and 48 h, respectively. b–d) In vitro UCL images of KB cells using the NaYF4 :Yb/Er UC HNSs as luminescent probe under excitation with 980 nm laser. All scale bars are 50 mm.
shows the in vitro cell viability of KB cells incubated with NaYF4 UC HNSs with different concentrations from 6.25 to 200 mg mL1 for 24 and 48 h, respectively. This result shows that more than 90 % of cell viabilities are observed, even at a high-dose concentration of 200 mg mL1 for the NaYF4 :Yb/Er UC HNSs after incubation for 24 and 48 h, respectively. Therefore, the UC HNSs are almost nontoxic to living cells and could be used as bioprobes for in vitro cell imaging or drug carriers for drug delivery.
Figure 3. Multicolor UC emissions tuning: a) room-temperature upconversion emission spectra of 1) Yb/Er (20/2 mol %), 2) Yb/Er (40/ 2 mol %), 3) Yb/Er (60/2 mol %), and 4) Yb/Tm (20/0.5 mol %) and Yb/ Tm/Er (20/0.5/0.2 mol %) co-doped NaYF4 UC HNSs in aqueous solutions (1 mg mL1), respectively. b) Their corresponding upconversion luminescent photographs taken under 980 nm NIR laser excitation with 2.0 s exposure time (laser power: 600 mW).
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The cellular uptake process by KB cells was examined by UCL microscopy. KB cells were incubated with NaYF4 :Yb/ Er UC HNSs (100 mg mL1) for 2 h and then washed with abundant phosphate-buffered saline (PBS) solution to remove the untouched nanomaterials. The luminescence images were recorded using an inverted florescence microscope equipped with an external 980 nm laser as the excitation source. As shown in Figure 4b–d, it can be seen that the cells incubated with UC HNSs show a strong green luminescence signal under 980 nm laser excitation (Figure 4c), and no autofluorescence background from the KB cells is observed. The overlay of the bright-field and UCL image in Figure 4d further indicates that the green emission is evident and part of the UC HNSs are internalized into the cells.[12] The above results demonstrate that the as-prepared
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NaYF4 :Yb/Er HNSs can be used as a good luminescent probe for cell imaging. Drug Loading/Release Properties and Examination of Therapeutic Effect Doxorubicin (DOX) was selected as a model drug to evaluate the loading and release behaviors of the NaYF4 UC HNSs. The concentration of DOX in the UC HNS-DOX composites could be determined by the characteristic absorption peak centered at 480 nm as shown in Figure 5a, and the successful loading of DOX onto the UC HNSs could be readily confirmed by a clear color change from white to red (Figure 5a, inset). We also systematically investigated the drug-loading behavior at three different pH values and
Figure 5. Drug loading/release behaviors and cancer-cell-killing capability investigation. a) A comparison of UV/Vis absorbance spectra of the UC HNSs, free DOX, and UC HNS-DOX at the loading value of pH 7.0. Inset: Color change before and after DOX attachment. b) Quantification of DOX loading capacity at different pH values. Higher drug loading quantity was achieved at higher pH value. c) DOX release curves in PBS medium at different pH values. d) Concentration-dependent cell viabilities of KB cells treated with free DOX and UC HNS-DOX composite. Error bars were calculated on the basis of six parallel samples. e–g) Inverted fluorescence microscopy images of KB cells incubated with UC HNS-DOX ([DOX] = 3 mg mL1) for 6 h. e) Bright-field image, f) fluorescent image, and g) overlay of (e) and (f).
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found that the amount of DOX loaded onto the UC HNSs is pH-dependent (Figure 5b). The saturated DOX-loading amount increased from 4 to 111 to 142 mg g1 as the pH values of the loading buffers increased from 5.5 to 7.0 to 8.0, respectively. The pH-dependent DOX-loading behavior could be explained by the enhanced intermolecular interaction between DOX and hollow PEI-UC HNSs owing to the deprotonation of NH2 groups of DOX molecules and dendrigraft cationic polymer PEI coated on the UC HNSs at higher pH values,[4b] and thus more and more DOX molecules could be adsorbed and stored in the inner cavity at higher pH values. To investigate the influence of UCL properties derived from drug loading, we collected the UCL spectra before and after DOX loading. The Supporting Information shows DOX absorption band overlaps with the green emission band of UC HNSs. Fluorescent resonant energy transfer (FRET) from UC HNSs to the attached DOX molecules possibly occurs, and thus 35 % green-emission intensity quenching is observed (Figure S6b, the Supporting Information). Figure 5c shows in vitro release profiles of DOX from the UC HNS-DOX in PBS solutions at two different pH values (7.4 and 5.0) at 37 8C. The drug-release kinetics show a clearly pH-dependent trend. Only 30 % of DOX was released from the UC HNS-DOX, even after 48 h in PBS solution at pH 7.4. In contrast, when the pH value was decreased to 5.0, the system showed a rapid drug-release rate that reached 38 % within 2 h, and more than 65 % of DOX was released after 48 h. The protonation of the amino group in the DOX molecule under acidic conditions would weaken its binding to the PEI polymer on the UC HNSs surface and accelerate the detachment of DOX from UC HNS-DOX.[11c] These pH-induced controllable drug-release systems could be used as a carrier for cancer chemotherapy in pathological sites owing to the pH of the microenvironment changing from 7.4 for normal tissues to acidic microenvironments for tumors.[13] In addition, we also collected the UC emission spectra as a function of the content of DOX release. Upon the release of DOX, the quenching effect was weakened and the emission intensity recovered gradually (Figure S6c, Supporting Information), which suggested that the nanocarriers could potentially serve as a probe for monitoring the drug-release efficiency during cancer therapy. Furthermore, to test the pharmacological activity of the UC HNS-DOX composites, the cytotoxic effect against KB cells was evaluated in vitro by means of a CCK-8 assay. Figure 5d shows the KB cell viabilities of free DOX and DOX-UC HNSs at different concentrations after 24 h incubation. It is noteworthy that the UC HNSDOX has much higher cell-killing ability than that of free DOX at nearly the same DOX concentration. The half-maximum inhibitory concentration (IC50) value for UC HNSDOX ( 3 mg mL1) was found to be much lower than that of free DOX ( 12.5 mg mL1), mainly owing to the greater cellular uptake as well as triggered drug release inside cells.[14] Figure 5e–g shows the inverted fluorescence images of KB cells after incubation with UC HNS-DOX (DOX concentration, 3 mg mL1) for 6 h. The strong red lumines-
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cence signal from DOX molecules is clear in Figure 5f and g, thereby confirming that the UC HNS-DOX composite could be internalized by the KB cells and then release DOX inside cells.[15] Therefore, the as-prepared UC HNSs are promising candidates for image-guided drug delivery.
Conclusion In summary, a facile one-pot template-free method is presented for the preparation of sub-100 nm NaYF4 UC HNSs by undergoing an Ostwald ripening mechanism. The main reaction solvent EG could assist the surfactant PEI to control the nucleation and growth of NaYF4 crystals, thereby producing sub-100 nm-scale HNSs. By doping different upconverters, multicolor UCL could be obtained under 980 nm NIR laser excitation. The hydrophilic PEI-coated products are of low toxicity to living cells and could be applied as bioprobes for UCL cell imaging in vitro. In addition, these HNSs can also be employed as nanocarriers for drug delivery. The loading and release of DOX from UC HNS-DOX could be controlled by varying pH values, which is favorable for drug delivery and controlled release to cancer cells. The inherent UCL from UC HNSs and fluorescence from DOX enable dual up- and downconversion fluorescent imaging to access the efficacy of the UC HNS-DOX drug-delivery system, thereby providing a potential platform for simultaneous imaging and therapy.
Experimental Section Materials PEI (Mw = 25 000) was purchased from Sigma–Aldrich. EG (A.R. grade), NH4F (96 %), YACHTUNGRE(NO3)3·x H2O (x 6; 99.9 %), YbACHTUNGRE(NO3)3·x H2O (x 5; 99.99 %), ErACHTUNGRE(NO3)3·x H2O (x 5; 99.9 %), TmACHTUNGRE(NO3)3·x H2O (x 5; 99.9 %), and HoACHTUNGRE(NO3)3·x H2O (x 5; 99.9 %) were all supplied by Alfa Aesar Reagent Company. DOX (99.9 %) was purchased from Beijing HuaFeng United Technology Co., Ltd. NaOH, HCl, and NaCl were purchased from Beijing Chemical Corporation and were analytical reagent grade. Deionized water was obtained with an 18 MW (SHRO-plus DI) system.
Synthesis of NaYF4 HNSs and NaYF4-Based UC HSs In a typical process, firstly, PEI (0.2 g) was dissolved in EG (20 mL) under stirring to obtain a transparent solution. Afterwards, a solution of Y3 + (0.5 mL, 2.0 m) was added. After mixing evenly, 8 mm of NH4F dissolved in EG (10 mL) was poured into the mixture solution. Finally, NaCl (1 mmol) was added into the mixture. After stirring for 15 min, the solution was transferred into a 45 mL autoclave. The system was sealed and treated at 200 8C for 12 h. The as-prepared precipitates were collected by centrifugation, washed several times with deionized water, and then the final products were obtained by freeze-drying. A similar process was used for preparing NaYF4-based UC HNSs by using a stoichiometric amount of YbACHTUNGRE(NO3)3 and ErACHTUNGRE(NO3)3/HoACHTUNGRE(NO3)3/TmACHTUNGRE(NO3)3 aqueous solutions instead of YACHTUNGRE(NO3)3 solution at the initial stage as described above.
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at 10 kV) and by means of transmission electron microscopy (TEM) (Tecnai G2 20 S-TWIN operated at 200 kV). X-ray diffraction (XRD) measurements were performed with a Japan Rigaku D/Max-2500 diffractometer with CuKa radiation (l = 1.54056 ). Fourier transform infrared (FTIR) spectra were measured with a Nicolet iN10 infrared microscope (Thermo scientific). N2 adsorption–desorption isotherms were carried out with a Micromeritics ASAP 2020M apparatus. Dynamic light scatting (DLS) analysis was performed with a Nicomp380 ZLS plus ZETADi. A Hitachi U-3900 spectrophotometer was used to obtain the UV/Vis absorption spectra. Upconversion luminescence (UCL) spectra were collected with a fluorescence spectrophotometer (Horiba Jobin Yvon FluoroLog3) using a 980 nm laser as the excitation source. Luminescent photographs were taken with a Nikon D3100 digital camera in a darkroom.
Cell Culture and In Vitro Cytotoxicity Estimation A human nasopharyngeal epidermal carcinoma cell line (KB cells) was cultured in a 25 cm2 flask (Corning Inc.) in a medium made up of RPMI1640 with l-glutamine supplemented with 10 % fetal bovine serum (FBS) at 37 8C under 5 % CO2. The suspension of KB cells was dispensed into 96-well plates at a density of 2 103 cells per well. After 24 h of incubation at 37 8C under a humidified atmosphere that contained 5 % CO2, each well was washed with PBS solution. Afterwards, different concentrations of hollow NaYF4 :Yb/Er UC HNSs were diluted with RPMI-1640 that contained l-glutamine and added into the 96-well plates. The cells were incubated with the hollow NaYF4 :Yb/Er UC HNSs for another 24 or 48 h at 37 8C with 5 % CO2, respectively. Finally, Cell Counting Kit-8 was added into each well and the cells were incubated for 1 h at 37 8C for cell-viability estimation. The absorbance of supernatants was measured at 450 nm with a microplate reader (SpectraMax M2 MDC, USA). For a test of cellular killing ability, KB cells were incubated with a series of concentrations of free DOX and UC HNS-DOX at a concentration of 0.75, 1.5, 3, 6, 12, 25, and 50 mg mL1 (DOX concentration) for 2 h and then changed to fresh culture medium after washing with PBS. After another 24 h incubation, the cell viabilities were also tested by CCK-8 assay.
Acknowledgements This work was supported by the National Natural Science Foundation of China (grant nos. 21101158, 21001108, 21177128, and 21171122) and the National Basic Research Programs of China (973 Program, nos. 2012CB932504 2011CB933403, and 2012CB934001).
Cell Imaging
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For UCL cell imaging, KB cells (105 per well) were grown on glass coverslips for 24 h. Subsequently, 1 mL of 100 mg mL1 NaYF4 :Yb/Er UC HNSs dispersed in RPMI-1640 with l-glutamine culture medium was added and incubated with cells for 2 h. After being washed with PBS three times, the cells were imaged with an inverted fluorescence microscope (Olympus IX71) equipped with a 980 nm laser (400 mW output power). DOX molecules with fluorescence could also be used as a bioprobe for cell imaging to examine the cellular uptake of UC HNS-DOX by KB cells. The KB cells were seeded in a culture plate and grown for 24 h, and then they were incubated with DOX-loaded hollow NaYF4-PEI UC HNSs (DOX = 3 mg mL1) at 37 8C for 6 h. Thereafter, the cells were rinsed with PBS for three times to remove free UC HNSs, fixed with formaldehyde (1 mL, 2.5 %) at 37 8C for 5 min, and then rinsed with PBS. Finally, PBS solution (1 mL) was added to the cells. The fixed cells were also imaged under an inverted fluorescence microscope.
Drug Loading and Releasing For the DOX-loading saturation experiment, NaYF4 UC HNSs (10 mg) were soaked in DOX solution (4 mL) with various concentrations from 50 to 3000 mm and stirred for 12 h at room temperature. To verify the effect of the pH value on DOX-loading efficiency, three different pH values at 5.5, 7.0, and 8.0 were chosen and investigated. The pH value of PBS medium was adjusted with 0.1 m NaOH and 0.1 m HCl. Free DOX was removed by centrifugation at 12 000 rpm for 10 min. The precipitated DOX-loaded UC HNSs (denoted as UC HNS-DOX) were washed two times with PBS and collected after freeze-drying. The DOX-loading efficiency could be calculated by the absorbance change of the supernatant before and after adsorption. For the DOX-release process, two identical UC HNS-DOX samples (50 mg) were immersed into PBS (10 mL) with two different pH values (pH 7.4 and 5.0), respectively, under slow shaking at 37 8C. At selected time intervals, PBS medium (3 mL) was removed for centrifugation. The released DOX concentration in the supernatant from the UC HNS-DOX was determined with a UV/Vis spectrophotometer at 480 nm, and then the supernatant was returned to the original release medium.
Characterization Morphologies of the hollow NaYF4 were obtained with a field-emission scanning electron microscope (FESEM) (S-4800, Hitachi, Japan, operated
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H. S. Wang, V. H. Y. Lee, S. H. Cheng, W. T. Wong, ACS Nano 2011, 5, 7838 – 7847; d) Y. L. Dai, X. J. Kang, D. M. Yang, X. J. Li, X. Zhang, C. X. Li, Z. Y. Hou. Z. Y. Cheng, P. A. Ma, J. Lin, Adv. Healthcare Mater. 2013, 2, 562 – 567. [13] a) J. V. Jokerst, T. Lobovkina, R. N. Zare, S. S. Gambhir, Nanomedicine 2011, 6, 715 – 728; b) X. X. Hu, Y. Wang, B. Peng, Chem. Asian J. 2013, 1, 319 – 327; c) W. W. Gao, J. M. Chan, O. C. Farokhzad, Mol. Pharm. 2010, 7, 1913 – 1920; d) G. Tian, Z. J. Gu, L. J. Zhou, W. Y. Yin, X. X. Liu, L. Yan, S. Jin, W. L. Ren, G. M. Xing, S. J. Li, Y. L. Zhao, Adv. Mater. 2012, 24, 1226 – 1231. [14] a) T. L. Doane, C. Burda, Chem. Soc. Rev. 2012, 41, 2885 – 2911; b) J. Shen, L. Zhao, G. Han, Adv. Drug Delivery Rev. 2013, 65, 744 – 755. [15] a) H. Meng, M. Liong, T. Xia, Z. X. Li, Z. X. Ji, J. I. Zink, A. E. Nel, ACS Nano 2010, 4, 4539 – 4550; b) J. N. Liu, W. B. Bu, L. M. Pan, J. L. Shi, Angew. Chem. Int. Ed. 2013, 52, 4375 – 4379; Angew. Chem. 2013, 125, 4471. Received: December 20, 2013 Revised: January 26, 2014 Published online: March 12, 2014
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One-Pot Template-Free Synthesis of NaYF4 Upconversion Hollow Nanospheres for Bioimaging and Drug Delivery Gan Tian,[a, c] Longsheng Duan,[a] Xiao Zhang,[a, b] Wenyan Yin,*[a] Liang Yan,[a] Liangjun Zhou,[a, d] Xiaodong Liu,[a] Xiaopeng Zheng,[a, d] Jinxia Li,[a] Zhanjun Gu,*[a] and Yuliang Zhao*[a, b, c] Abstract: Hollow-structured nanomaterials with fluorescent properties are extremely attractive for image-guided cancer therapy. In this paper, sub100 nm and hydrophilic NaYF4 upconversion (UC) hollow nanospheres (HNSs) with multicolor UC luminescence and drug-delivery properties were successfully prepared by a facile one-pot template-free hydrothermal route using polyetherimide (PEI) polymer as the stabilizing agent. XRD,
SEM, TEM, and N2-adsorption/desorption were used to characterize the asobtained products. The growth mechanism of the HNSs has been systematically investigated on the basis of the Ostwald ripening. Under 980 nm excitation, UC emissions of HNSs can be Keywords: drug delivery · hollow nanospheres · luminescence · onepot synthesis · Ostwald ripening
Introduction
used as luminescent probes for biological imaging or as tracers for drug-release monitoring.[1c] Lanthanide (Ln)-doped upconversion (UC) luminescent nanomaterials, which can convert low-energy (near-infrared (NIR) photon) excitation to higher-energy (UV-visible light) emission by means of multiple absorption or energy transfer,[2] have been increasingly proposed as next-generation bioprobes for bioimaging since they have many advantages over conventional organic dye markers and quantum dots, such as high physicochemical stability, low toxicity, reduced photodamage to living organisms, high signal-to-noise ratio, and strong tissue penetration ability.[2b] Therefore, the design and fabrication of hollow UC nanostructures would undoubtedly be of great importance in the field of image-guided drug delivery.[2d] Generally, a template-assisted strategy that involves the coating of templates with nanocrystals (precursors or targeted products) and then removal of the templates has been widely employed for the preparation of hollow-structured UC nanomaterials since the templating method offers important advantages, including easy operation and well-defined structural features of the products with narrow size distribution.[3] Nonetheless, the disadvantages of this method, such as tedious synthetic procedures and low yields, have largely impeded the upscaled production and largescale applications. Moreover, collapse of some fraction of the hollow structures always occurs during the templatecore removal process through high-temperature calcination (e.g., carbon spheres) or chemical dissolution (e.g., using HF to etch silica spheres). Unfortunately, the products obtained from the high-temperature calcination processes com-
Recently, luminescent hollow-structured materials have attracted considerable attention owing to their unique properties and potential applications in biomedical fields.[1] Relative to conventional biomedical systems, these novel multifunctional materials not only provide a cavity structure for the controlled storage/delivery of therapeutic drugs but also possess luminescence properties, which means they can be
[a] Dr. G. Tian,+ Dr. L. Duan,+ Dr. X. Zhang, Dr. W. Yin, Dr. L. Yan, Dr. L. Zhou, Dr. X. Liu, Dr. X. Zheng, Dr. J. Li, Dr. Z. Gu, Prof. Y. Zhao CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics Chinese Academy of Sciences, Beijing 100049 (P.R. China) E-mail: [email protected] [email protected] [email protected] [b] Dr. X. Zhang, Prof. Y. Zhao National Center for Nanosciences and Technology of China Beijing, 100190 (P.R. China) [c] Dr. G. Tian,+ Prof. Y. Zhao College of Chemistry, Sichuan University Chengdu, 610064 (P.R. China) [d] Dr. L. Zhou, Dr. X. Zheng College of Materials Science and Opto-electronic Technology Graduate University of Chinese Academy of Sciences Beijing 100049 (P.R. China) [+] These authors contributed equally. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201301695.
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tuned by a simple change of the concentration or combination of various upconverters. As a result, the PEIcoated HNSs could be used as efficient probes for in vitro upconversion luminescence (UCL) cell imaging. Furthermore, a doxorubicin storage/release behavior and cancer-cell-killing ability investigation reveal that the product has the potential to be a drug carrier for cancer therapy.
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pletely lose the chemical functional groups located on their water solubility and good biocompatibility show intense surfaces, thereby resulting in poor water solubility and thus multicolor upconversion luminescence (UCL) under 980 nm depriving their prospects in the biological field.[3b] AlternaNIR laser excitation. Considering the presence of the cavity tively, to simplify the preparation procedure, a template-free in the hollow nanostructures, the material can be used as an process has also been developed for the construction of effective drug carrier when using doxorubicin (DOX) as hollow UC structures.[4] Zhao et al. reported a facile solua model drug. The drug-loading and release properties, cytotion-phase synthesis of NaYF4 :Yb/Er(Tm) UC hollow toxicity, and therapeutic effects are investigated in detail. spheres based on the nanoscale growth induced by the Kirkendall effect.[4a] Lin et al. employed the same strategy for controllable synthesis of NaYF4 :Yb/Er hollow spheres[4b] Results and Discussion and core–shell-structured Yb(OH)CO3@YbPO4 :Er hollow Morphology and Structure microspheres.[4c] However, all these synthetic schemes require the use of their corresponding oxides or carbonates as Large-scaled NaYF4 nanospheres can be achieved by means the precursor. Therefore, it is still significant and urgent to of hydrothermal treatment at 200 8C for 12 h using PEI as explore other template-free strategies that aim toward the surfactant. As can be seen in Figure 1a, these nanoa facile one-step synthetic approach for obtaining hollow spheres with a size of (50 6) nm (Figure S1, Supporting Instructures. Currently, utilization of the well-known classical formation) have a rugged and bumpy surface (Figure S2, physical phenomenon known as the Ostwald ripening mechSupporting Information). The broken nanospheres are hemianism provides an optional opportunity for one-pot synthespherical or bowl-like in shape, which suggests the presence sis of Ln-based luminescent hollow structures.[5] This desiraof a characteristic cavity in hollow structures; this could also be confirmed by the TEM image presented in Figure 1a, ble strategy simplifies the synthetic procedure and improves inset. The N2 adsorption–desorption isotherm in Figure 1b the production yield.[5a] So far, one-pot fabrication of hollow UC structures that undergo the Ostwald ripening mechashows that the isotherm is type II and reveals the existence nism has been lingering far behind the significant progress of mesoporosity in the sample owing to the presence of hysthat has been made in the template-synthesis method. In adteresis in the adsorption.[5c] The Brunauer–Emmett–Teller dition, the as-prepared products are usually microscaled, (BET) surface area is about 20.02 m2 g1, and the pore [5c] which makes them unsuitable for biological applications. volume is 0.126 cm3 g1 as calculated from N2 isotherms at As a result, developing a facile strategy based on the Ost77 K. The pore-size distribution (Figure 1b, inset) was calcuwald ripening mechanism to fabricate sub-100 nm and highly water-soluble UC hollow nanospheres (HNSs) is still a big challenge. In this work, we report an efficient, one-pot route to synthesize sub-100 nm water-soluble NaYF4-based UC HNSs under hydrothermal conditions based on a gas-bubble-assisted Ostwald ripening mechanism from solid spheres to hollow spheres.[6] In this reaction system, we used ethylene glycol (EG) as the main solvent and polyethylenimine (PEI) as the structure-stabilizing agent. The solvent EG with abundant hydroxyl groups could also be seen as the second stabilizing agent to assist PEI for efficiently controlling the nucleation and growth of the NaYF4 crystal, thereby largely decreasing the size of the self-assembled Figure 1. Materials characterization: a) SEM image, inset: a typical TEM image showing the hollow structures; solid spheres and thus achieving b) N adsorption–desorption isotherm, inset: pore-size distribution curve; c) XRD patterns; and d) FTIR spec2 nanoscaled HNSs. The PEI- trum of the as-obtained NaYF4 hollow nanospheres, inset: photograph of the as-prepared PEI-coated NaYF4 coated UC HNSs with high HNSs dispersed in deionized (DI) water (1 mg mL1).
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lated using the Barrett–Joyner–Halenda (BJH) distribution from the adsorption branch, thus indicating the pore-size distributions in the mesoporous (2–50 nm) and macroporous (> 50 nm) regions with a maximum peak pore diameter of approximately 35 nm. The XRD pattern in Figure 1c indicates a high crystallinity of the as-prepared nanoparticles. The cubic NaYF4 phase (JCPDS no. 77-2042) is the dominant phase, but a small quantity of hexagonal NaYF4 phase (JCPDS no. 16-0334) is also observed. Previous reports have demonstrated the stabilizing agent PEI could strongly coordinate with Y3 + ions, which would decrease both the nucleation process and nuclei growth and thus suppress the formation of hexagonal NaYF4 phase.[7] Nevertheless, the prolonged heat treatment benefits the phase transformation from the metastable cubic phase to the more stable hexagonal phase.[8] The presence of PEI polymer on the surface of the HNSs was confirmed by FTIR spectroscopy as shown in Figure 1d. The bands at 2929 and 2852 cm1 are attributed to the stretching vibrations of the CH2 group, respectively. In addition, peaks centered at 1636 and 1431 cm1 are internal vibrations of the amide group, and the peak at 1533 cm1 is the characteristic asymmetric vibration of NH2 group,[4b, 7a] thus demonstrating the existence of the PEI polymer. Owing to the presence of hydrophilic PEI on the surface, these nanospheres could be readily dispersed in deionized water, thus forming a nearly transparent aqueous solution (Figure 1d, inset).
needed for structure evolution from a solid to hollow structure by means of an Ostwald ripening process is up to at least 24 h.[9] In the present work, HNSs can be obtained within 12 h. The formation process can be explained by the possible reaction processes proposed by Equations (1–4) in this system, whereby the gas bubbles generated under high temperature and pressure would play an important role in the formation of HNSs.[6c] YNO3 þ PEI ! Y3þ @PEI complex þ 3 NO3 4 NH4 F þ NaCl þ 3 NO3 þ Y3þ @PEI ! NaYF4 @PEI þNH4 Cl þ 3 NH4 NO3 D
NH4 Cl ! NH3 " þHCl " D
NH4 NO3 ! N2 O " þ2 H2 O
ð2Þ ð3Þ ð4Þ
In this reaction system, the surfactant PEI has good affinity towards Y3 + and will form a Y3 + @PEI complex at the very beginning [Eq. (1)].[7a] An appropriate PEI amount is indispensable for the formation of NaYF4 HNSs.[5c] In a series of control groups, we find that PEI-free products are bulk polyhedron and predominantly irregular nanoparticles. Also, amounts of PEI that exceed 0.20 g do not benefit the formation of HNSs but produce solid spheres with smooth surfaces (Figure S3, Supporting Information). Owing to the low free Y3 + concentration in the solution that arises from the weak dissociation tendency of the Y-PEI complex,[4b] the slow nucleation of NaYF4 [Eq. (2)] is the rate-determining step. Simultaneously, a large amount of gas bubbles could be generated in the reaction system [Eqs. (3) and (4)] and serve as nucleation centers for the freshly formed nuclei.[6b] These smaller NaYF4 nuclei would assemble and form the solid NaYF4 spheres to minimize the interfacial energy.[5c, 9b] These kinds of metastable solid spheres can be obtained in large quantities by adjusting the reaction time to 2 h (Figure 2b). Relative to previously reported work,[5, 6] the self-assembled solid spheres here are much smaller. In our procedure, we used an EG/H2O system as the reaction media, the addition of a small amount of H2O is crucial for the oriented aggregation of NaYF4 nuclei (Figure S4, Supporting Information) and benefits the Ostwald ripening process.[10] EG with abundant hydroxyl groups could also coordinate with Y3 + ions and thus influence the NaYF4 nucleation and selfassembly process, thereby causing the formation of solid spheres with nanoscale size. Products prepared in parallel without EG are predominantly bulk prisms (Figure S5, Supporting Information). As the hydrothermal process progresses, the system energy is high enough to trigger the Ostwald ripening process.[6c, 9a] During this period, smaller-sized, less crystalline, and higher-surface-energy nuclei in the inner cores will be gradually dissolved and merged with nanoparticles in the outer surface, thereby producing channels that connect the inner and outer spaces in the NaYF4 shells (Figure 2c). Finally, after further prolonging the reaction to 12 h, the cores within the hollow spheres were consumed com-
The Proposed Formation Mechanism The possible formation process of NaYF4 UC HNSs can be explained by the Ostwald ripening process; the whole process is illustrated in Figure 2a. Usually, the reaction time
Figure 2. Proposed formation mechanism: a) schematic illustration of the proposed mechanism for the formation of NaYF4 HNSs by means of Ostwald ripening. TEM images of NaYF4 products at different growth times: b) 2, c) 6, and d) 12 h, respectively. e) Time-dependent XRD patterns of the products.
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tributed to 4H11/2 !4I15/2 and 4S3/2 !4I15/2 transitions of Er3 + ions, respectively, whereas the red band that peaked at 653 nm is ascribed to an 4F9/2 !4I15/2 transition.[2a] By varying the Yb3 + doping level, the color output from green to yellow could be realized. Increasing the amount of Yb3 + dopants decreased the Yb···Er interatomic distance and thus facilitated back-energy transfer from Er3 + to Yb3 + , thereby decreasing the transition possibility of green (4H11/2, 4S3/2 ! 4 I15/2) light emissions and increasing the ratio of red to green emission.[3b, 7b] For Yb/Tm co-doped HNSs, six characteristic bands centered at 361, 450, 475, 650, 725, and 800 nm belong to the 1D2 !3H6, 1D2 !3F4, 1G4 !3H6, 1G4 !3F4, 3F3 ! 3 H6, and 3H4 !3H6 transitions of Tm3 + ions, respectively,[2b] and the sample exhibits a blue emission (Figure 3b, (4)). In the dual-emitter dopant system, characteristic peaks that belong to each emitter can be clearly observed (Figures 3a, (4) and (5)) and a nearly white-light emission (Figures 3b, (4) and (5)) is achieved in the dual-emission process.
pletely and hollow nanospheres formed (Figure 2d). The XRD patterns in Figure 2e show that thermodynamically more stable hexagonal-phase NaYF4 appeared, and the relative intensity increased in a time-dependent manner, partly supporting the dissolution–recrystallization involved in the Ostwald ripening process, whereby the thermodynamically more stable structure would be preferentially generated during the recrystallization process.[5c] UCL Properties NaYF4 is considered one of the most efficient host matrixes for UC nanomaterials owing to its low phonon frequency, which can minimize nonradiative losses and maximize the radiative emission intensity.[2d, 8c] The UCL of upconverterdoped samples can be clearly observed under the excitation of a 980 nm continual-wave laser, and the color output could be manipulated by adjusting the combination or concentration of the doped upconverters. The UCL spectra and their corresponding photographs of Yb/Er, Yb/Tm, or Yb/ Er,Tm co-doped NaYF4 HNSs are given in Figure 3. For the Yb/Er co-doped HNSs, the weak blue emission band that peaked at 410 nm can be assigned to an 2H9/2 !4I15/2 transition, and the green bands centered at 521 and 541 nm are at-
Cell Viability and UCL Cell Imaging Cytotoxicity is an essential concern when it comes to the development of nanomaterials for biomedical imaging and cancer-therapy applications.[11] The viability of KB cells after exposure to NaYF4 :Yb/Er UC HNSs was measured by a standard Cell Counting Kit-8 (CCK-8) assay. Figure 4a
Figure 4. Cytotoxicity test and UCL cell imaging: a) Cell viabilities of KB cells incubated with NaYF4 :Yb/Er UC HNSs at different concentrations for 24 and 48 h, respectively. b–d) In vitro UCL images of KB cells using the NaYF4 :Yb/Er UC HNSs as luminescent probe under excitation with 980 nm laser. All scale bars are 50 mm.
shows the in vitro cell viability of KB cells incubated with NaYF4 UC HNSs with different concentrations from 6.25 to 200 mg mL1 for 24 and 48 h, respectively. This result shows that more than 90 % of cell viabilities are observed, even at a high-dose concentration of 200 mg mL1 for the NaYF4 :Yb/Er UC HNSs after incubation for 24 and 48 h, respectively. Therefore, the UC HNSs are almost nontoxic to living cells and could be used as bioprobes for in vitro cell imaging or drug carriers for drug delivery.
Figure 3. Multicolor UC emissions tuning: a) room-temperature upconversion emission spectra of 1) Yb/Er (20/2 mol %), 2) Yb/Er (40/ 2 mol %), 3) Yb/Er (60/2 mol %), and 4) Yb/Tm (20/0.5 mol %) and Yb/ Tm/Er (20/0.5/0.2 mol %) co-doped NaYF4 UC HNSs in aqueous solutions (1 mg mL1), respectively. b) Their corresponding upconversion luminescent photographs taken under 980 nm NIR laser excitation with 2.0 s exposure time (laser power: 600 mW).
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The cellular uptake process by KB cells was examined by UCL microscopy. KB cells were incubated with NaYF4 :Yb/ Er UC HNSs (100 mg mL1) for 2 h and then washed with abundant phosphate-buffered saline (PBS) solution to remove the untouched nanomaterials. The luminescence images were recorded using an inverted florescence microscope equipped with an external 980 nm laser as the excitation source. As shown in Figure 4b–d, it can be seen that the cells incubated with UC HNSs show a strong green luminescence signal under 980 nm laser excitation (Figure 4c), and no autofluorescence background from the KB cells is observed. The overlay of the bright-field and UCL image in Figure 4d further indicates that the green emission is evident and part of the UC HNSs are internalized into the cells.[12] The above results demonstrate that the as-prepared
Wenyan Yin, Zhanjun Gu, Yuliang Zhao et al.
NaYF4 :Yb/Er HNSs can be used as a good luminescent probe for cell imaging. Drug Loading/Release Properties and Examination of Therapeutic Effect Doxorubicin (DOX) was selected as a model drug to evaluate the loading and release behaviors of the NaYF4 UC HNSs. The concentration of DOX in the UC HNS-DOX composites could be determined by the characteristic absorption peak centered at 480 nm as shown in Figure 5a, and the successful loading of DOX onto the UC HNSs could be readily confirmed by a clear color change from white to red (Figure 5a, inset). We also systematically investigated the drug-loading behavior at three different pH values and
Figure 5. Drug loading/release behaviors and cancer-cell-killing capability investigation. a) A comparison of UV/Vis absorbance spectra of the UC HNSs, free DOX, and UC HNS-DOX at the loading value of pH 7.0. Inset: Color change before and after DOX attachment. b) Quantification of DOX loading capacity at different pH values. Higher drug loading quantity was achieved at higher pH value. c) DOX release curves in PBS medium at different pH values. d) Concentration-dependent cell viabilities of KB cells treated with free DOX and UC HNS-DOX composite. Error bars were calculated on the basis of six parallel samples. e–g) Inverted fluorescence microscopy images of KB cells incubated with UC HNS-DOX ([DOX] = 3 mg mL1) for 6 h. e) Bright-field image, f) fluorescent image, and g) overlay of (e) and (f).
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found that the amount of DOX loaded onto the UC HNSs is pH-dependent (Figure 5b). The saturated DOX-loading amount increased from 4 to 111 to 142 mg g1 as the pH values of the loading buffers increased from 5.5 to 7.0 to 8.0, respectively. The pH-dependent DOX-loading behavior could be explained by the enhanced intermolecular interaction between DOX and hollow PEI-UC HNSs owing to the deprotonation of NH2 groups of DOX molecules and dendrigraft cationic polymer PEI coated on the UC HNSs at higher pH values,[4b] and thus more and more DOX molecules could be adsorbed and stored in the inner cavity at higher pH values. To investigate the influence of UCL properties derived from drug loading, we collected the UCL spectra before and after DOX loading. The Supporting Information shows DOX absorption band overlaps with the green emission band of UC HNSs. Fluorescent resonant energy transfer (FRET) from UC HNSs to the attached DOX molecules possibly occurs, and thus 35 % green-emission intensity quenching is observed (Figure S6b, the Supporting Information). Figure 5c shows in vitro release profiles of DOX from the UC HNS-DOX in PBS solutions at two different pH values (7.4 and 5.0) at 37 8C. The drug-release kinetics show a clearly pH-dependent trend. Only 30 % of DOX was released from the UC HNS-DOX, even after 48 h in PBS solution at pH 7.4. In contrast, when the pH value was decreased to 5.0, the system showed a rapid drug-release rate that reached 38 % within 2 h, and more than 65 % of DOX was released after 48 h. The protonation of the amino group in the DOX molecule under acidic conditions would weaken its binding to the PEI polymer on the UC HNSs surface and accelerate the detachment of DOX from UC HNS-DOX.[11c] These pH-induced controllable drug-release systems could be used as a carrier for cancer chemotherapy in pathological sites owing to the pH of the microenvironment changing from 7.4 for normal tissues to acidic microenvironments for tumors.[13] In addition, we also collected the UC emission spectra as a function of the content of DOX release. Upon the release of DOX, the quenching effect was weakened and the emission intensity recovered gradually (Figure S6c, Supporting Information), which suggested that the nanocarriers could potentially serve as a probe for monitoring the drug-release efficiency during cancer therapy. Furthermore, to test the pharmacological activity of the UC HNS-DOX composites, the cytotoxic effect against KB cells was evaluated in vitro by means of a CCK-8 assay. Figure 5d shows the KB cell viabilities of free DOX and DOX-UC HNSs at different concentrations after 24 h incubation. It is noteworthy that the UC HNSDOX has much higher cell-killing ability than that of free DOX at nearly the same DOX concentration. The half-maximum inhibitory concentration (IC50) value for UC HNSDOX ( 3 mg mL1) was found to be much lower than that of free DOX ( 12.5 mg mL1), mainly owing to the greater cellular uptake as well as triggered drug release inside cells.[14] Figure 5e–g shows the inverted fluorescence images of KB cells after incubation with UC HNS-DOX (DOX concentration, 3 mg mL1) for 6 h. The strong red lumines-
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cence signal from DOX molecules is clear in Figure 5f and g, thereby confirming that the UC HNS-DOX composite could be internalized by the KB cells and then release DOX inside cells.[15] Therefore, the as-prepared UC HNSs are promising candidates for image-guided drug delivery.
Conclusion In summary, a facile one-pot template-free method is presented for the preparation of sub-100 nm NaYF4 UC HNSs by undergoing an Ostwald ripening mechanism. The main reaction solvent EG could assist the surfactant PEI to control the nucleation and growth of NaYF4 crystals, thereby producing sub-100 nm-scale HNSs. By doping different upconverters, multicolor UCL could be obtained under 980 nm NIR laser excitation. The hydrophilic PEI-coated products are of low toxicity to living cells and could be applied as bioprobes for UCL cell imaging in vitro. In addition, these HNSs can also be employed as nanocarriers for drug delivery. The loading and release of DOX from UC HNS-DOX could be controlled by varying pH values, which is favorable for drug delivery and controlled release to cancer cells. The inherent UCL from UC HNSs and fluorescence from DOX enable dual up- and downconversion fluorescent imaging to access the efficacy of the UC HNS-DOX drug-delivery system, thereby providing a potential platform for simultaneous imaging and therapy.
Experimental Section Materials PEI (Mw = 25 000) was purchased from Sigma–Aldrich. EG (A.R. grade), NH4F (96 %), YACHTUNGRE(NO3)3·x H2O (x 6; 99.9 %), YbACHTUNGRE(NO3)3·x H2O (x 5; 99.99 %), ErACHTUNGRE(NO3)3·x H2O (x 5; 99.9 %), TmACHTUNGRE(NO3)3·x H2O (x 5; 99.9 %), and HoACHTUNGRE(NO3)3·x H2O (x 5; 99.9 %) were all supplied by Alfa Aesar Reagent Company. DOX (99.9 %) was purchased from Beijing HuaFeng United Technology Co., Ltd. NaOH, HCl, and NaCl were purchased from Beijing Chemical Corporation and were analytical reagent grade. Deionized water was obtained with an 18 MW (SHRO-plus DI) system.
Synthesis of NaYF4 HNSs and NaYF4-Based UC HSs In a typical process, firstly, PEI (0.2 g) was dissolved in EG (20 mL) under stirring to obtain a transparent solution. Afterwards, a solution of Y3 + (0.5 mL, 2.0 m) was added. After mixing evenly, 8 mm of NH4F dissolved in EG (10 mL) was poured into the mixture solution. Finally, NaCl (1 mmol) was added into the mixture. After stirring for 15 min, the solution was transferred into a 45 mL autoclave. The system was sealed and treated at 200 8C for 12 h. The as-prepared precipitates were collected by centrifugation, washed several times with deionized water, and then the final products were obtained by freeze-drying. A similar process was used for preparing NaYF4-based UC HNSs by using a stoichiometric amount of YbACHTUNGRE(NO3)3 and ErACHTUNGRE(NO3)3/HoACHTUNGRE(NO3)3/TmACHTUNGRE(NO3)3 aqueous solutions instead of YACHTUNGRE(NO3)3 solution at the initial stage as described above.
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at 10 kV) and by means of transmission electron microscopy (TEM) (Tecnai G2 20 S-TWIN operated at 200 kV). X-ray diffraction (XRD) measurements were performed with a Japan Rigaku D/Max-2500 diffractometer with CuKa radiation (l = 1.54056 ). Fourier transform infrared (FTIR) spectra were measured with a Nicolet iN10 infrared microscope (Thermo scientific). N2 adsorption–desorption isotherms were carried out with a Micromeritics ASAP 2020M apparatus. Dynamic light scatting (DLS) analysis was performed with a Nicomp380 ZLS plus ZETADi. A Hitachi U-3900 spectrophotometer was used to obtain the UV/Vis absorption spectra. Upconversion luminescence (UCL) spectra were collected with a fluorescence spectrophotometer (Horiba Jobin Yvon FluoroLog3) using a 980 nm laser as the excitation source. Luminescent photographs were taken with a Nikon D3100 digital camera in a darkroom.
Cell Culture and In Vitro Cytotoxicity Estimation A human nasopharyngeal epidermal carcinoma cell line (KB cells) was cultured in a 25 cm2 flask (Corning Inc.) in a medium made up of RPMI1640 with l-glutamine supplemented with 10 % fetal bovine serum (FBS) at 37 8C under 5 % CO2. The suspension of KB cells was dispensed into 96-well plates at a density of 2 103 cells per well. After 24 h of incubation at 37 8C under a humidified atmosphere that contained 5 % CO2, each well was washed with PBS solution. Afterwards, different concentrations of hollow NaYF4 :Yb/Er UC HNSs were diluted with RPMI-1640 that contained l-glutamine and added into the 96-well plates. The cells were incubated with the hollow NaYF4 :Yb/Er UC HNSs for another 24 or 48 h at 37 8C with 5 % CO2, respectively. Finally, Cell Counting Kit-8 was added into each well and the cells were incubated for 1 h at 37 8C for cell-viability estimation. The absorbance of supernatants was measured at 450 nm with a microplate reader (SpectraMax M2 MDC, USA). For a test of cellular killing ability, KB cells were incubated with a series of concentrations of free DOX and UC HNS-DOX at a concentration of 0.75, 1.5, 3, 6, 12, 25, and 50 mg mL1 (DOX concentration) for 2 h and then changed to fresh culture medium after washing with PBS. After another 24 h incubation, the cell viabilities were also tested by CCK-8 assay.
Acknowledgements This work was supported by the National Natural Science Foundation of China (grant nos. 21101158, 21001108, 21177128, and 21171122) and the National Basic Research Programs of China (973 Program, nos. 2012CB932504 2011CB933403, and 2012CB934001).
Cell Imaging
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For UCL cell imaging, KB cells (105 per well) were grown on glass coverslips for 24 h. Subsequently, 1 mL of 100 mg mL1 NaYF4 :Yb/Er UC HNSs dispersed in RPMI-1640 with l-glutamine culture medium was added and incubated with cells for 2 h. After being washed with PBS three times, the cells were imaged with an inverted fluorescence microscope (Olympus IX71) equipped with a 980 nm laser (400 mW output power). DOX molecules with fluorescence could also be used as a bioprobe for cell imaging to examine the cellular uptake of UC HNS-DOX by KB cells. The KB cells were seeded in a culture plate and grown for 24 h, and then they were incubated with DOX-loaded hollow NaYF4-PEI UC HNSs (DOX = 3 mg mL1) at 37 8C for 6 h. Thereafter, the cells were rinsed with PBS for three times to remove free UC HNSs, fixed with formaldehyde (1 mL, 2.5 %) at 37 8C for 5 min, and then rinsed with PBS. Finally, PBS solution (1 mL) was added to the cells. The fixed cells were also imaged under an inverted fluorescence microscope.
Drug Loading and Releasing For the DOX-loading saturation experiment, NaYF4 UC HNSs (10 mg) were soaked in DOX solution (4 mL) with various concentrations from 50 to 3000 mm and stirred for 12 h at room temperature. To verify the effect of the pH value on DOX-loading efficiency, three different pH values at 5.5, 7.0, and 8.0 were chosen and investigated. The pH value of PBS medium was adjusted with 0.1 m NaOH and 0.1 m HCl. Free DOX was removed by centrifugation at 12 000 rpm for 10 min. The precipitated DOX-loaded UC HNSs (denoted as UC HNS-DOX) were washed two times with PBS and collected after freeze-drying. The DOX-loading efficiency could be calculated by the absorbance change of the supernatant before and after adsorption. For the DOX-release process, two identical UC HNS-DOX samples (50 mg) were immersed into PBS (10 mL) with two different pH values (pH 7.4 and 5.0), respectively, under slow shaking at 37 8C. At selected time intervals, PBS medium (3 mL) was removed for centrifugation. The released DOX concentration in the supernatant from the UC HNS-DOX was determined with a UV/Vis spectrophotometer at 480 nm, and then the supernatant was returned to the original release medium.
Characterization Morphologies of the hollow NaYF4 were obtained with a field-emission scanning electron microscope (FESEM) (S-4800, Hitachi, Japan, operated
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