Gd)F4:Yb3+,Er3+ Composite

Jan 17, 2014 - Porous hydroxyapatite (HAp) composite fibers functionalized with up-conversion (UC) luminescent and magnetic Na(Y/Gd)F4:Yb3+,Er3+ ...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/Langmuir

Multifunctional Hydroxyapatite/Na(Y/Gd)F4:Yb3+,Er3+ Composite Fibers for Drug Delivery and Dual Modal Imaging Min Liu,† Hui Liu,§ Shufen Sun,† Xuejiao Li,‡ Yanmin Zhou,*,† Zhiyao Hou,*,‡ and Jun Lin*,‡ †

Department of Periodontology, Stomatological Hospital, Jilin University, Changchun 130021, P. R. China State Key laboratory of Rare Earth Resource utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China § Department of Human Anatomy, College of Basic Medical Sciences, Jilin University, Changchun 130021, P. R. China ‡

S Supporting Information *

ABSTRACT: Porous hydroxyapatite (HAp) composite fibers functionalized with up-conversion (UC) luminescent and magnetic Na(Y/Gd)F4:Yb3+,Er3+ nanocrystals (NCs) have been fabricated via electrospinning. After transferring hydrophobic oleic acid-capped Na(Y/Gd)F4:Yb3+,Er3+ NCs into aqueous solution, these water-dispersible NCs were dispersed into precursor electrospun solution containing CTAB. Na(Y/Gd)F4:Yb3+,Er3+@ HAp composite fibers were fabricated by the high temperature treatment of the electrospun Na(Y/Gd)F4:Yb3+,Er3+ NCs decorated precursor fibers. The biocompatibility test on MC 3T3-E1 cells using MTT assay shows that the HAp composite fibers have negligible cytotoxity, which reveals the HAp composite fibers could be a drug carrier for drug delivery. Because the contrast brightening is enhanced at increased concentrations of Gd3+, the HAp composite fibers can serve as T1 magnetic resonance imaging contrast agents. In addition, the composites uptaken by MC 3T3-E1 cells present the UC luminescent emission of Er3+ under the excitation of a 980 nm near-infrared laser. The above findings reveal Na(Y/ Gd)F4:Yb3+,Er3+@HAp composite fibers have potential applications in drug storage/release and magnetic resonance/UC luminescence imaging. release behaviors.11 However, many biological tissues and drug molecules present autofluorescence under UV radiation, which greatly decrease the signal-to-noise and detection sensitivity. Porous and DCL HAp materials are unsuited for severing as fluorescent labels for bioimaging. Lanthanide-based upconversion luminescence (UCL) materials, which emit higher-energy visible emission after being excited by low-energy NIR light,12−15 have many fascinating features including weak autofluorescence background,16−18 strong penetration ability, less photodamage to cells, high signal-to-noise ratio, and detection sensitivity.19−23 NaYF4:Yb3+,Er3+ nanocrystals (NCs) decorated SiO2 composite fibers with porous structure and UCL emission of Er3+ under 980 nm laser excitation were fabricated by electrospinning, and their properties of anticancer drug storage and UCL cell imaging were studied.24 The dual modal imaging, which possesses the advantages of combines the high spatial and temporal resolution from UCL labels and the temporal resolution from MRI contrast agents,25,26 can avoid the limitations of each single imaging modality. In addition, HAp-based materials have more solubility and less toxicity than silica-based materials. Therefore, the rational design for developing multifunctional HAp materials with

1. INTRODUCTION Hydroxyapatite [HAp, Ca10(PO4)6(OH)2] as bioceramics material has attracted more attention for bone substitute materials due to the excellent osteoconductive property and the similar composition to bone mineral.1 With the fascinating features of stable porous structure, −OH active bonds on the surface, bioactivity, nontoxicity, and noninflammatory properties, porous HAp with various morphologies and surface properties have also been considered as drug delivery systems (DDSs) for loading and releasing of pharmaceutical molecules.2−4 Compared with the traditional DDSs based on polymer materials,5 porous HAp materials can be stored drugs without adding other pressure. Meanwhile, the porous HAp materials combining luminescent and magnetic properties in one DDS not only possess the similar drug storage/release properties to the other inorganic porous drug carriers but also own luminescent and magnetic properties which can be served as biological labels for high sensitivity/resolution fluorescent imaging and magnetic resonance imaging (MRI). Porous HApbased DDSs functionalized with luminescent and magnetic properties present potential applications in diagnosis and treatment of diseases.6−11 We have fabricated fiber-like and belt-like porous HAp:Eu3+ down-conversion luminescence (DCL) materials, mainly focusing on their luminescent properties and drug storage/ © 2014 American Chemical Society

Received: January 15, 2014 Published: January 17, 2014 1176

dx.doi.org/10.1021/la500131d | Langmuir 2014, 30, 1176−1182

Langmuir

Article

RIMC)/OIMC] × 100%. IMC-UCMNCs@HAp was immersed in 5 mL of phosphate buffer solution (PBS, 10 mM, pH = 7.4). At certain time intervals, 100 μL of PBS supernatant was taken out by centrifugation, and an equal volume of fresh PBS was added again for the continued drug release. The concentrations of the released IMC in the supernatant solutions were recorded by UV−vis measurement (monitored on 320 nm). Measurement of TNF-α by ELISA. MC cells were plated in 24well plates (5 × 104 cells per well) for incubating overnight at 37 °C. Then, the media with different amounts of the free AGEs (0, 25, 50, 100, 200, and 400 μg mL−1) and (AGEs + IMC-UCMNCs@HAp) were added to each well for incubating another 24 h at 37 °C. The amount of IMC-UCMNCs@HAp was 100 μg mL−1 for each well with the different amounts of AGEs. After collecting the supernatant from each well, the mixture of 20 μL of the supernatant and 180 μL of 0.05 M carbonate buffer (pH = 9.6) was added in the each well of the 96-well ELISA plates and then maintained at 4 °C overnight. After removing the above solution, 200 μL of 1 wt % BSA in the PBS (pH = 7.4) was added in each well and then maintained at 37 °C for 1.5 h. The above solution was removed, and then the diluted rabbit anti-rat TNF-α polyclonal antibody (1:1000, 1 wt % BSA solution) was added into wells, 100 μL for each, followed by incubation at 37 °C for 2 h. Then the 96-well ELISA plates were immediately washed 3 times with the PBST solution (pH = 7.4, phosphoric acid buffer 8 mM, NaCl 140 mM, KCl 2.6 mM, Tween-20 0.07%). 100 μL of the diluted HRP-labeled goat anti-rabbit antibody (1:1000, 1 wt % BSA solution) was added into each well, followed by incubation at 37 °C for 1 h. The 96-well ELISA plates were immediately washed 3 times with the PBST solution (pH = 7.4) again and patted dry. Subsequently, 100 μL of chromogenic substrate (pH = 5.0, citric acid buffer 0.1 M, TMB 5 mg L−1, hydrogen peroxide 0.006%) was added into each well for 15 min incubation in the dark, followed by adding 50 μL of stop buffer (sulfuric acid, 3 M) to end the reaction. Finally, the OD values were recorded under a microplate reader (Thermo Multiskan mk3) at 450 nm. UCL Imaging of UCMNCs@HAp. 5 × 104 MC-3T3-E1 cells per well were plated on a clean coverslip, putting in each well of the 6-well culture plates for incubation overnight as a monolayer. Then the media with a certain concentration of ultrasonically treated UCMNCs@HAp were added to each well, and the cells were incubated at 37 °C for 3 h. The above solution was removed, and the 6-well culture plates were washed three times with PBS. The cells were fixed with 2.5% formaldehyde at 37 °C for 10 min and then washed three times with PBS again. UCL imaging was performed with a converted upconversion luminescence microscopy (UCLM) that rebuilt on an inverted florescence microscope (Nikon Ti−S), an infrared laser excited unit (FF735-Di01-25×36, Nikon), and laser diode driver (KS3-11312-312, BWT). UCMNCs@HAp uptake by cells was excited by infrared laser (FF735-Di01-25×36, Nikon) at 980 nm (BWT Beijing LTD, China) with output power of 250 mW. In Vitro Magnetic Resonance Imaging of UCMNCs@HAp. The in vitro T1-weighted MR images were performed with a 0.5 T MRI magnet (Shanghai Niumag Corporation Ration NM120-Analyst). The dilutions of the utrasonically treated UCMNCs@HAp dispersing in deionized water with various Gd concentrations (0.25, 0.5, 1, 2, and 5 mM) were loaded in 2 mL tubes for T1 measurements. The slope of the curve that plotting as relaxation time 1/T1 (s−1) vs molar concentration of Gd (mM) was determined as the relaxivity values of r1. Characterization. A D8 Focus diffractometer (Bruker) was used to record the X-ray diffraction (XRD) patterns for confirming the phase purity and crystallization of the products. The U-3310 spectrophotometer was used to record the UV−vis adsorption spectra. The field emission scanning electron microscope (Philips XL 30) was used to examine the morphologies and dimensions of the samples. A FEI Tecnai G2 S-Twin transmission electron microscope was used to inspect the transmission electron microscopy (TEM) micrographs of the samples. Nitrogen adsorption/desorption measurements with specific surface area and pore volume of composites were tested through a Micromeritics ASAP 2020 M apparatus. The UC emission

porous structure and UCL/magnetic properties as drug carrier is undoubtedly of great importance in the field of drug delivery and multimodal imaging. Gd3+/Yb3+/Er3+ codoped NaYF4 NCs combine the UCL and magnetic properties due to the 4f7 electronic configuration of Er3+ and Gd3+. As a continuation of our former research, in this paper we fabricate Na(Y/Gd)F4:Yb3+,Er3+ NCs (denoted as UCMNCs) decorated porous HAp composite fibers via the electrospinning method. Since the development of electrospinning in 1934,27 this method is an effective and simple technique for preparing fibers from a rich variety of materials.28−32 The porous Na(Y/Gd)F4:Yb3+,Er3+@HAp (denoted as UCMNCs@HAp) composite fibers were obtained after high temperature annealing of the hybrid precursor fibers containing UCMNCs. After being fully characterized, the composite fibers were studied as a DDS by using Indomethacin (IMC, anti-inflammatory drug) as a model drug. The drug loading/release properties, cell cytotoxicity, cellular uptake behavior, and UCL/MR dual modal imaging were also examined.

2. EXPERIMENTAL SECTION Synthesis of Water-Dispersible α-Na(Y/Gd)F4:Yb3+,Er3+NCs. The rare earth oleate complexes were synthesized according to a literature method for the preparation of iron oleate complex.33 Our previous methodology for the synthesis of NaYF4:Yb3+,Er3+ NCs was adopted to prepare oleic acid-capped Na(Y/Gd)F4:Yb3+,Er3+ NCs (72% Y/8% Gd/17% Yb/3% Er).24 The obtained oleic acid stabilized UCMNCs were dispersed in 0.5 mL of chloroform (CHCl3) and then added dropwise into 5 mL of aqueous solution containing 0.1 g of cetyltrimethylammonium bromide (CTAB). The above mixing solution was vigorously stirred at 60 °C to induce evaporation of the CHCl3.34 Fabrication of UCMNCs@HAp Composite Fibers. (NH4)H2PO4 and Ca(NO3)2·4H2O were added into water−ethanol solution (v/v = 1:6, pH = 2−3) containing CTAB (1.5 wt % in the mixture) and water-dispersible UCMNCs under magnetic stirring. PVP (8.5 wt %) was added into above solution to form electrospun sol after magnetic stirring for 3 h. The electrospun parameters of preparing UCMNCs decorated precursor fibers, such as the distance between the spinneret and collector, the value of high voltage, and the spinning rate, were set according to our previous electrospinning process.11 Finally, the porous UCMNCs@HAp composite fibers were obtained after annealing UCMNCs decorated precursor fibers at 600 °C in air with the heating rate of 1 °C min−1 and maintained at this temperature for 3 h. Porous UCMNCs@HAp were fragmentated with ultrasonics before drug loading and cellular uptake. Biocompatibility of the UCMNCs@HAp Composite Fibers. MTT cell assay was used to estimate the cell cytotoxicity of the UCMNCs@HAp composite fibers on the MC-3T3-E1 cell. MC-3T3E1 cells were plated in a 96-well plate (5000 cells per well) for incubating 24 h, and then the media with different amounts of UCMNCs@HAp (ultrasonically treated) were added to each well. After incubating another 24 h, 5 mg mL−1 MTT solution (20 μL per well) was added into the 96-well plate for incubating 4 h. After removing the supernatant and adding 150 μL of DMSO into each well, the 96-well plate was measured via a microplate reader (Thermo Multiskan mk3) at 490 nm. In Vitro Drug Loading/Release. 100 mg of UCMNCs@HAp (ultrasonically treated) was dispersed in 10 mL of the ethanol−water (4:1, v/v) mixed solution containing 15 mg of IMC (Aladdin Chemistry Co., Ltd.). After soaking for 24 h with stirring, the IMCloaded UCMNCs@HAp was separated by centrifugation and denoted as IMC-UCMNCs@HAp. The residual IMC content (RIMC) of supernatant solutions and the original IMC content (OIMC) were obtained by UV−vis absorption spectra at a wavelength of 320 nm, and the IMC-loading efficiency can be calculated as follows: [(OIMC − 1177

dx.doi.org/10.1021/la500131d | Langmuir 2014, 30, 1176−1182

Langmuir

Article

Figure 1. TEM and HR-TEM (inset) images of water-soluble α-Na(Y/Gd)F4:Yb3+,Er3+ NCs (a), XRD pattern of α-Na(Y/Gd)F4:Yb3+,Er3+ NCs, and JCPDS card 06-0342 of α-NaYF4 (b), and UC emission spectrum of α-Na(Y/Gd)F4:Yb3+,Er3+ NCs excited by 980 nm NIR laser (c) with its luminescent photograph (inset). spectra were carried out on the optical parametric oscillator (Continuum Sunlite) with a 980 nm laser as the excitation source and detected by a R955 (Hamamatsu). Up-conversion luminescence images were observed digitally by a Nikon multiple CCD camera (DSRi1).

3. RESULTS AND DISCUSSION Physical and Chemical Characterization of Nanocrystals and Composites. Figure 1a displays the respective TEM image of water-dispersible Na(Y/Gd)F4:Yb3+,Er3+ NCs. In order to fulfill the requirement of electrospun precursor sol, the oleic acid-capped Na(Y/Gd)F4:Yb3+,Er3+ NCs were transferred into aqueous phase through employing CTAB as a secondary surfactant. The UCMNCs dispersing in water present well monodispersity with narrow size distribution (average diameter around 5 nm). The lattice fringes (d = 0.31 nm for 111 plane of cubic phase NaYF4) revealing the crystalline of UCMNCs can be observed clearly from HR-TEM image (inset of Figure 1a). The XRD result (Figure 1b) confirms that all the diffraction peaks for UCMNCs coincides well with the literature values of cubic phase NaYF4 (JCPDS No. 06-0342). Under 980 nm laser excitation, UCMNCs emit red light (the inset of Figure 1c) ascribed to 2H11/2/4S3/2 → 4I15/2 (from 510 to 575 nm) and 4 H9/2 → 4I15/2 (660 and 675 nm) electron transitions of Er3+ ions (Figure 1C).35 The XRD patterns of the pure HAp fibers (a) and UCMNCs@HAp composite fibers (b) are presented in Figure 2. Well-defined diffraction peaks can be assigned to hexagonal phase of HAp with JCPDS card No. 09-043 (Figure 2a), suggesting that the electrospun precursor composites have crystallized into HAp after 600 °C annealing. When UCMNCs decorated electrospun precursor samples are calcined at 600 °C (Figure 2b), the emergence of new sharp diffraction peaks (comparison with pure HAp samples) is assigned to the hexagonal structured NaYF4 (JCPDS No. 06-0342). The results demonstrate cubic phase NaYF4 is transformed to the hexagonal phase in the UCMNCs@HAp composite fibers after annealing treatment. The phase transition of NaYF4 from

Figure 2. XRD patterns for the pure HAp fibers (a) and UCMNCs@ HAp composite fibers (b) as well as the standard JCPDS cards 090432 of HAp and 16-0334 of β-NaYF4 for comparison.

cubic phase to hexagonal phase is the exothermic process, and the hexagonal phase is thermodynamically stable compared with the cubic phase.33,36,37 In our fabrication of UCMNCs@ HAp composite fibers, the phase transformation of NaYF4 occurs after the electrospun precursor composites are calcined at 600 °C. Figure 3 presents the SEM images of the UCMNCs decorated precursor fibers and UCMNCs@HAp composite fibers, respectively. The as-fabricated precursor fibers are uniform with length of several tens to hundred micrometers and diameters ranging from 0.2 to 1.0 μm (Figure 3a). After being annealed at high temperature to remove the organic components, UCMNCs@HAp composite fibers with the diameters of 75−200 nm were obtained (Figure 3b). The TEM technique was used to characterize the porous structure of UCMNCs@HAp composite fibers and shown in Figure 4a. UCMNCs@HAp still presents fiber-like morphology, and the diameter shrunk to about 100 nm. Owing to the different electron penetrability, the porous structure of UCMNCs@HAp composite fibers is easily recognizable. From the HR-TEM image (Figure 4b), the lattice fringes with the distance of 0.28 nm (well consistent with the d211 value of the hexagonal phase 1178

dx.doi.org/10.1021/la500131d | Langmuir 2014, 30, 1176−1182

Langmuir

Article

HAp) are clearly visible. The result reveals the crystalline of hexagonal phase HAp after being annealed at 600 °C. The CTAB has an important effect during the formation of porous HAp due to the electrostatic interaction between the cationic surfactant CTAB and PO43− in the electrospun solution. After forming a metal citrate complex in the metal−salt solution, these complexes interacted electrostatically with the CTAB micelles to form CTAB−metal citrate complexes in the solution containing CTAB-stabilized UCMNCs. Then adding PVP to adjust viscoelasticity of the above solution, CTAB−metal citrate complexes, CTAB micelles, and UCMNCs can exist in the electrospun hybrid sol. The porous structures of UCMNCs@ HAp was formed after high-temperature treatment of precursor fibers to remove the CTAB template.11,38 The UC emission spectrum of the UCMNCs@HAp composite fibers via 980 nm NIR laser excitation is shown in Figure S1a. Green emission bands ranging from 510 to 570 nm are attributed to 4 H11/2/4S3/2 → 4I15/2 of Er3+, and red emission bands at 630− 700 nm are assigned to 4F9/2 → 4I15/2 of Er3+. The UC mechanisms and the energy transfer process of Yb3+/Er3+ codoped materials are schematically shown in Figure S1b. Drug Loading/Releasing Properties and Anti-inflammatory Efficacy. The respective N2 adsorption/desorption isotherms of UCMNCs@HAp and IMC−UCMNCs@HAp composite fibers are presented in Figure S2. UCMNCs@HAp and IMC−UCMNCs@HAp show similar N2 adsorption/ desorption isotherms with the typical H1-hysteresis loops, demonstrating the presence of porous and less influence on the pore structure after loading IMC. The BET surface area, pore volume, and average pore size of UCMNCs@HAp are about 25.5 m2/g, 0.152 cm3/g, and 12.5 nm, respectively. The BET surface area, pore volume, and average pore size of IMC− UCMNCs@HAp sharply reduced compared with UCMNCs@ HAp due to the loading of IMC. The FT-IR spectra of the UCMNCs@HAp and IMC− UCMNCs@HAp samples are presented in Figure S3. In Figure S3a, for the pure UCMNCs@HAp, the absorption bands at 3430 and 1640 cm−1 are ascribed to the O−H vibration which indicates that OH groups are present on the surface of UCMNCs@HAp. The 1468 and 1428 cm−1 peaks are from CO32− groups of water. The peaks at 1095, 1042, and 965 cm−1 can be attributed to ν3 antisymmetric stretching and ν1 nondegenerated symmetric stretching of P−O bond, respectively. The peaks at 606 and 565 cm−1 can be related to the ν4 vibration of O−P−O bond, and the peaks ranging from 450 to 475 cm−1 may be assigned to ν2 bending of the O−P−O bond.39 For the IMC−UCMNCs@HAp (Figure S3b), the absorption bands ranging from 1295 to 1645 cm−1 can be detected, which confirms that the IMC is loaded into the porous UCMNCs@HAp successfully. During the drug storage process, the IMC molecules can be adsorbed into the pores of UCMNCs@HAp composite fibers, and the loading content of IMC in IMC-UCMNCs@HAp is 12.6 wt % determined by TG analysis. During the IMC release process, the PBS enters into the pores of IMC-UCMNCs@ HAp; the IMC then slowly diffuses from the system along the solvent-filled pores. The IMC release profile from IMC− UCMNCs@HAp in PBS is presented in Figure 5. 39 wt % of IMC is released from IMC−UCMNCs@HAp within 1 h; the initial burst release may be ascribed to the free IMC desorption from the outer surface of IMC−UCMNCs@HAp. Then about 83 wt % of IMC is leached from IMC−UCMNCs@HAp within 24 h, and the release of IMC can be continued more than 36 h.

Figure 3. SEM images of the UCMNCs decorated precursor fibers (a) and 600 °C annealing derived UCMNCs@HAp composite fibers (b).

Figure 4. TEM (a) and HR-TEM (b) images of UCMNCs@HAp composite fibers.

1179

dx.doi.org/10.1021/la500131d | Langmuir 2014, 30, 1176−1182

Langmuir

Article

results suggest that the IMC releasing from IMC-UCMNCs@ HAp effectively attenuated the secretion of TNF-α from the MC cells induced by AGEs. in Vitro MR and UCL Imaging. The biocompatibility of biomaterials is very important; the lanthanides-based UC luminescent materials show no or low cytotoxicity.46−48 The ultrasonically treated UCMNCs@HAp composite fibers were incubated with MC 3T3-E1 cells, and the cell cytotoxicity of UCMNCs@HAp was evaluated by MTT assay. As seen from Figure S4, the MC 3T3-E1 cells viabilities showed no significant changes; more than 95% cells viability was detected under the varying concentration range (1.5625−100 μg/mL). Based on the above results, UCMNCs@HAp composite fibers show good biocompatibility and can serve as drug carriers for biomedical applications. Nutrients and foreign substances can be taken up by cells via endocytosis, and endocytosis can be divided into phagocytosis and pinocytosis. Cellar uptake of large particles is defined as phagocytosis, and pinocytosis is used to internalize fluid containing substances surrounding the cells.49 As seen from the TEM image of ultrasonically treated UCMNCs@HAp composite fibers (Figure S5a), the diameter of fiber fragments is about 100 nm and the length of fiber fragments ranges from 200 to 400 nm. Figure S5b gives out the dynamic light scattering data; the size of fiber fragments ranges from 60 nm to 0.9 μm (both length and diameter). The UCMNCs@HAp composite fibers (the length with several hundred micrometers) can be cut into short fragments effectively by the ultrasonic treatment. After ultrasonically treating, the size of fiber fragments mainly belongs to large particles (as shown in Figure S5b), the fiber fragments as foreign substances derived via phagocytosis are enclosed within phagosomes and then fuse with lysosomes. To evaluate the MR properties of UCMNCs@HAp, an in vitro T1-weighted MR imaging for UCMNCs@HAp was conducted on a Shanghai Niumag Corporation Ration NM120-Analyst system. As shown in Figure 7, from the

Figure 5. Cumulative release curve of IMC leaching from IMC− UCMNCs@HAp system in the PBS buffer.

The sustained release of the rest of IMC can be attributed to the interaction between IMC and the UCMNCs@HAp. In order to verify the anti-inflammatory efficacy of IMC release from IMC-UCMNCs@HAp, MC cells were induced with advanced glycation end products (AGEs) and measured the expressions of tumor necrosis factor alpha in cultured media. Cytokines, such as interleukin 1-beta (IL-1β), interleukin 6 (IL-6), interleukin 1-alpha (IL-1α), and tumor necrosis factor alpha (TNF-α), are the major pro-inflammatory factors responsible for early inflammatory responses.40,41 AGEs were synthesized irreversibly by the nonenzymatic glycation of proteins, which can be observed to accumulate with aging in various organs.42,43 The expressions of IL-1β, IL-6, IL-1α, and TNF-α can be triggered by the receptor for AGEs.44,45 AGEsinduced inflammatory is associated with increased production of pro-inflammatory cytokines. The effects of MC cells on AGEs-induced TNF-α production with various amounts of AGEs (0−400 μg/mL) were examined, and the results are presented in Figure 6. AGEs were used to promote the

Figure 6. IMC-UCMNCs@HAp inhibits pro-inflammatory cytokine production (TNF-α) in AGEs-induced MC cells (*P < 0.05).

Figure 7. Plot of R1 (1/T1, s−1) versus Gd3+ concentration (5, 2, 1, 0.5, and 0.25 mM). The slope indicates the specific relativity. Inset: the T1weighted and color-mapped MR images of UCMNCs@HAp with deionized water (0 mM) as the reference.

secretion of TNF-α in MC cells, and the expression of TNF-α in MC cells was elevated significantly with the increasing amounts of AGEs by comparison with the control group. In an investigation of the anti-inflammatory effect of IMCUCMNCs@HAp (100 μg/mL) on AGEs-induced MC cells, pro-inflammatory cytokine production (TNF-α) was measured via ELISA. IMC releasing from IMC-UCMNCs@HAp decreased the secretion levels of TNF-α in cultured media at all concentrations of AGEs compared to the control group. The

slope of the plot of 1/T1 versus the Gd3+ concentration, the ionic longitudinal relaxivity (R1) was determined to be 0.179 mM−1 s−1. With the increase of Gd3+ concentration, the T1weighted MRI resulted in brighter images (inset of Figure 7). To obtain a clear view of the dose-dependent positive enhancement effect, colored T1-weighted MR images are also presented (inset of Figure 7). As the concentration increased, 1180

dx.doi.org/10.1021/la500131d | Langmuir 2014, 30, 1176−1182

Langmuir

Article

HAp (b) composite fibers (Figure S3); cell viability of MC 3T3-E1 cells incubated with the various concentrations of UCMNCs@HAp composite fibers for 24 h (Figure S4); TEM image (a) and size distribution (b) of UCMNCs@HAp fiber fragments (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

the color of the MR images changed from blue to yellow, corresponding to the signal changes from low to high level. The results above demonstrated that the composite fibers could serve as T1-MRI contrast agent. Up-conversion luminescent microscopy (UCLM) was used to detect the interaction of MC 3T3-E1 cells with the UCMNCs@HAp under NIR laser excitation at 980 nm. Figure 8 shows the inverted fluorescence

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (Y.Z.). *E-mail [email protected] (Z.H.). *E-mail [email protected] (J.L.). Notes

The authors declare no competing financial interest.

■

Figure 8. Inverted florescence microscope images of MC 3T3-E1 cells incubated with UCMNCs@HAp composite fibers: bright-field image (a), UC luminescent image (b), and the overlay of bright-field and UC luminescent images (c). Scale bars for all images are 30 μm.

ACKNOWLEDGMENTS This project is financially supported by the National Natural Science Foundation of China (NSFC 51202239, 51332008, 51172228) and National Basic Research Program of China (2014CB643803).

■

microscopy images of MC 3T3-E1 cells incubated with UCMNCs@HAp (50 μg mL−1) for 4 h. The green fluorescence signals can be detected via the inverted fluorescence microscopy under the excitation of 980 nm laser (Figure 8b). The overlay of bright-field (Figure 8a) and fluorescence images confirms that the green UC luminescence signals are evident in the intracellular region (Figure 8c). The result of UC luminescence image demonstrates that UCMNCs@HAp fiber fragments can penetrate the cell membrane of MC 3T3-E1 cells and indicates that UCMNCs@HAp composite fiber is potential candidate for serving as fluorescence probe for cell imaging.

4. CONCLUSIONS Magnetic/UC luminescent UCMNCs@HAp composite fibers were obtained after high temperature treatment of Na(Y/ Gd)F 4 :Yb 3+ ,Er 3+ NCs decorated precursor fibers. The UCMNCs@HAp composite fibers present porous structure, regular fiber-like morphology, and low cell cytotoxicity, which may be further served as carriers for the delivery of drugs, protein, genes, or siRNA. Under 980 nm laser excitation, the UCMNCs@HAp displays typical emission bands owing to the electron transitions of Er3+ ions. The UCMNCs@HAp composite fibers owing UC luminescent properties, the interaction of cells with UCMNCs@HAp can be directly detected by up-conversion luminescence microscopy without labeling other fluorescent materials. Because of the positive signal-enhancement ability of Gd3+ ions, the UCMNCs@HAp composite fibers can be served as T1 MRI contrast agents. Electrospinning is an effective route for the fabrication of multifunctionalized HAp composite fibers. Meanwhile, UCMNCs@HAp composite fibers can be also used as contrast agents for UCL and MR dual-modality imaging.

■

REFERENCES

(1) Hench, L. L.; Wilson, J. Surface-active biomaterials. Science 1984, 226, 630−636. (2) Itokazu, M.; Yang, W.; Aoki, T.; Ohara, A.; Kato, N. Synthesis of antibiotic-loaded interporous hydroxyapatite blocks by vacuum method and in vitro drug release testing. Biomaterials 1998, 19, 817−819. (3) Rauschmann, M. A.; Wichelhaus, T. A.; Stirnal, V.; Dingeldein, E.; Zichner, L.; Schnettler, R.; Alt, V. Nanocrystalline hydroxyapatite and calcium sulfate as biodegradable composite carrier material for local delivery of antibiotics in bone infections. Biomaterials 2005, 26, 2677−2684. (4) Almirall, A.; Larrecq, G.; Delgado, J. A. A. Fabrication of low temperature macroporous hydroxyapatite scaffolds by foaming and hydrolysis of an alpha-TCP paste. Biomaterials 2004, 17, 3671−3680. (5) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M. Polymeric systems for controlled drug release. Chem. Rev. 1999, 99, 3181−3198. (6) Lebugle, A.; Pelle, F.; Charvillat, C.; Rousselot, I.; Chane-Ching, J. Y. Colloidal and monocrystalline Ln(3+) doped apatite calcium phosphate as biocompatible fluorescent probes. Chem. Commun. 2006, 42, 606−608. (7) Yang, P. P.; Quan, Z. W.; Li, C. X.; Kang, X. J.; Lian, H. Z.; Lin, J. Bioactive, luminescent and mesoporous europium-doped hydroxyapatite as a drug carrier. Biomaterials 2008, 29, 4341−4347. (8) Chen, F.; Huang, P.; Zhu, Y. J.; Wu, J.; Cui, D. X. Multifunctional Eu3+/Gd3+ dual-doped calcium phosphate vesicle-like nanospheres for sustained drug release and imaging. Biomaterials 2012, 33, 6447−6455. (9) Mondéjar, S. P.; Kovtun, A.; Epple, M. Lanthanide-doped calcium phosphate nanoparticles with high internal crystallinity and with a shell of DNA as fluorescent probes in cell experiments. J. Mater. Chem. 2007, 17, 4153−4159. (10) Zhang, C. M.; Li, C. X.; Huang, S. S.; Hou, Z. Y.; Cheng, Z. Y.; Yang, P. P.; Peng, C.; Lin, J. Self-activated luminescent and mesoporous strontium hydroxyapatite nanorods for drug delivery. Biomaterials 2010, 31, 3374−3383. (11) Hou, Z. Y.; Yang, P. P.; Lian, H. Z.; Wang, L. L.; Zhang, C. M.; Li, C. X.; Chai, R. T.; Cheng, Z. Y.; Lin, J. Luminescent porous silica fibers as drug carriers. Eur. Chem. J. 2009, 15, 6973−6982. (12) Haase, M.; Schäfer, H. Upconverting nanoparticles. Angew. Chem., Int. Ed. 2011, 50, 5808−5829. (13) Abel, K. A.; Boyer, J. C.; van Veggel, F. C. J. M. Hard proof of the NaYF4/NaGdF4 nanocrystal core/shell structure. J. Am. Chem. Soc. 2009, 131, 14644−14645.

ASSOCIATED CONTENT

S Supporting Information *

UC emission spectrum of UCMNCs@HAp composite fibers excited by 980 nm NIR laser (a), schematic illustration for the excitation, energy transfer, and UC luminescence processes of the Yb3+/Er3+ codoped luminescent materials (b) (Figure S1); N2 adsorption/desorption isotherms of UCMNCs@HAp (a) and IMC−UCMNCs@HAp (b) composite fibers (Figure S2); FT-IR spectra of UCMNCs@HAp (a) and IMC−UCMNCs@ 1181

dx.doi.org/10.1021/la500131d | Langmuir 2014, 30, 1176−1182

Langmuir

Article

(14) Boyer, J. C.; Cuccia, L. A.; Capobianco, J. A. Synthesis of colloidal upconverting NaYF4:Er3+/Yb3+ and Tm3+/Yb3+ monodisperse nanocrystals. Nano Lett. 2007, 7, 847−852. (15) Wang, F.; Deng, R. R.; Wang, J.; Wang, Q. X.; Han, Y.; Zhu, H. M.; Chen, X. Y.; Liu, X. G. Nat. Mater. 2011, 10, 968−973. (16) Wang, L. Y.; Yan, R. X.; Hao, Z. Y.; Wang, L.; Zeng, J. H.; Bao, J.; Wang, X.; Peng, Q.; Li, Y. D. Tuning upconversion through energy migration in core-shell nanoparticles. Angew. Chem., Int. Ed. 2005, 44, 6054−6057. (17) Zhang, F.; Braun, G. B.; Shi, Y. F.; Zhang, Y. C.; Sun, X. H.; Reich, N. O.; Zhao, D. Y.; Stucky, G. Fabrication of Ag@SiO2@ Y2O3:Er nanostructures for bioimaging: Tuning of the upconversion fluorescence with silver nanoparticles. J. Am. Chem. Soc. 2010, 132, 2850−2851. (18) Wang, F.; Liu, X. G. Recent advances in the chemistry of lanthanide-doped upconversion nanocrystals. Chem. Soc. Rev. 2009, 38, 976−989. (19) Wang, F.; Banerjee, D.; Liu, Y. S.; Chen, X. Y.; Liu, X. G. Upconversion nanoparticles in biological labeling, imaging, and therapy. Analyst 2010, 135, 1839−1854. (20) Chen, Z. G.; Chen, H. L.; Hu, H.; Yu, M. X.; Li, F. Y.; Zhang, Q.; Zhou, Z. G.; Yi, T.; Huang, C. H. Versatile synthesis strategy for carboxylic acid-functionalized upconverting nanophosphors as biological labels. J. Am. Chem. Soc. 2008, 130, 3023−3029. (21) Shen, J.; Sun, L. D.; Yan, C. H. Luminescent rare earth nanomaterials for bioprobe applications. Dalton Trans. 2008, 42, 5687−5697. (22) Nyk, M.; Kumar, R.; Ohulchanskyy, T. Y.; Bergey, E. J.; Prasad, P. N. High contrast in vitro and in vivo photoluminescence bioimaging using near infrared to near infrared up-conversion in Tm3+ and Yb3+ doped fluoride nanophosphors. Nano Lett. 2008, 8, 3834−3838. (23) Wang, C.; Tao, H. Q.; Cheng, L.; Liu, Z. Near-infrared light induced in vivo photodynamic therapy of cancer based on upconversion nanoparticles. Biomaterials 2011, 32, 6145−6154. (24) Hou, Z. Y.; Li, C. X.; Ma, P. A.; Cheng, Z. Y.; Li, X. J.; Zhang, X.; Dai, Y. L.; Yang, D. M.; Lian, H. Z.; Lin, J. Up-conversion luminescent and porous NaYF4:Yb3+, Er3+@SiO2 nanocomposite fibers for anti-cancer drug delivery and cell imaging. Adv. Funct. Mater. 2012, 22, 2713−2722. (25) Johnson, N. J. J.; Oakden, W.; Stanisz, G. J.; Prosser, R. S.; van Veggel, F. C. J. M. Size-tunable, ultrasmall NaGdF4 nanoparticles: Insights into their T-1 MRI contrast enhancement. Chem. Mater. 2011, 23, 3714−3722. (26) Yang, H.; Zhuang, Y.; Hu, H.; Du, X.; Zhang, C.; Shi, X.; Wu, H.; Yang, S. Silica-coated manganese oxide nanoparticles as a platform for targeted magnetic resonance and fluorescence imaging of cancer cells. Adv. Funct. Mater. 2010, 20, 1733−1741. (27) Formhals, A. US Patent Specification 1 975 504, 1934. (28) Madhugiri, S.; Dalton, A.; Gutierrez, J.; Ferraris, J. P.; Balkus, K. J. Electrospun MEH-PPV/SBA-15 composite nanofibers using a dual syringe method. J. Am. Chem. Soc. 2003, 125, 14531−14538. (29) Yao, L.; Haas, T. W.; Guiseppi-Elie, A.; Bowlin, G. L.; Simpson, D. G.; Wnek, G. E. Electrospinning and stabilization of fully hydrolyzed poly(vinyl alcohol) fibers. Chem. Mater. 2003, 15, 1860− 1864. (30) Wu, J.; Coffer, J. L. Emissive erbium-doped silicon and germanium oxide nanofibers derived from an electrospinning process. Chem. Mater. 2007, 19, 6266−6276. (31) Ge, J. J.; Hou, H.; Li, Q.; Graham, M. J.; Greiner, A.; Reneker, D. H.; Harris, F. W.; Cheng, S. Z. D. Assembly of well-aligned multiwalled carbon nanotubes in confined polyacrylonitrile environments: Electrospun composite nanofiber sheets. J. Am. Chem. Soc. 2004, 126, 15754−15761. (32) Sill, T. J.; von Recum, H. A. Electrospinning: Applications in drug delivery and tissue engineering. Biomaterials 2008, 29, 1989− 2006. (33) Park, J.; An, K. J.; Hwang, Y. S.; Park, J. G.; Noh, H. J.; Kim, J. Y.; Park, J. H.; Hwang, N. M.; Hyeon, T. Ultra-large-scale syntheses of monodisperse nanocrystals. Nat. Mater. 2004, 3, 891−895.

(34) Kim, J.; Lee, J. E.; Lee, J.; Yu, J. H.; Kim, B. C.; An, K.; Hwang, Y.; Shin, C. H.; Park, J. G.; Kim, J.; Hyeon, T. Magnetic fluorescent delivery vehicle using uniform mesoporous silica spheres embedded with monodisperse magnetic and semiconductor nanocrystals. J. Am. Chem. Soc. 2006, 128, 688−689. (35) Wang, X.; Zhuang, J.; Peng, Q.; Li, Y. D. A general strategy for nanocrystal synthesis. Nature 2005, 437, 121−124. (36) Lee, J. S.; Kim, Y. J. The effects of preparation conditions on the structural and up-conversion properties of NaYF4:Yb3+, Er3+ nano powders. Opt. Mater. 2010, 33, 1111−1115. (37) Wei, Y.; Lu, F. Q.; Zhang, X. R.; Chen, D. P. Synthesis and characterization of efficient near-infrared upconversion Yb and Tm codoped NaYF4 nanocrystal reporter. J. Alloys Compd. 2007, 427, 333−340. (38) Yao, J.; Tjandra, W.; Chen, Y. Z.; Tam, K. C.; Ma, J.; Soh, B. Hydroxyapatite nanostructure material derived using cationic surfactant as a template. J. Mater. Chem. 2003, 13, 3053−3057. (39) Doat, A.; Fanjul, M.; Pellé, F.; Hollande, E.; Lebugle, A. Europium-doped bioapatite: a new photostable biological probe, internalizable by human cells. Biomaterials 2003, 24, 3365−3371. (40) Giovanni, G.; Paola, A.; Alberto, M. Cytokines as a key component of cancer-related inflammation. Cytokine 2008, 43, 374− 379. (41) Geng, J.; Xiao, S. Y.; Zheng, Z. H.; Song, S. Y.; Zhang, L. R. Gambogic acid protects from endotoxin shock by suppressing proinflammatory factors in vivo and in vitro. Inflamm. Res. 2013, 62, 165− 172. (42) Verzijl, N.; De Groot, J.; Oldehinkel, E.; Bank, R. A.; Thorpe, S. R.; Baynes, J. W.; Bayliss, M. T.; Bijlsma, J. W.; Lafeber, F. P.; Tekoppele, J. M. Agerelated accumulation of Maillard reaction products in human articular cartilage collagen. Biochem. J. 2000, 350, 381−387. (43) De Groot, J. The AGE of the matrix: chemistry, consequence and cure. Curr. Opin. Pharmacol. 2004, 4, 301−305. (44) Rasheed, Z.; Akhtar, N.; Haqqi, T. M. Advanced glycation end products induce the expression of interleukin-6 and interleukin-8 by receptor for advanced glycation end product-mediated activation of mitogen-activated protein kinases and nuclear factor-κB in human osteoarthritis chondrocytes. Rheumatology 2011, 50, 838−851. (45) Chen, Y. J.; Sheu, M. L.; Tsai, K. S.; Yang, R. S.; Liu, S. H. Advanced glycation end products induce peroxisome proliferatoractivated receptor γ down-regulation-related inflammatory signals in human chondrocytes via toll-like receptor-4 and receptor for advanced glycation end products. PLoS One 2013, 8, e66611. (46) Ferrand, A. C.; Imbert, D.; Chauvin, A. S.; van Dervyver, C. D. B.; Büzli, J. C. G. Non-cytotoxic, bifunctional Eu-III and Tb-III luminescent macrocyclic complexes for luminescence resonant energytransfer experiments. Chem.Eur. J. 2007, 13, 8678−8687. (47) Xiong, L. Q.; Chen, Z. G.; Yu, M. X.; Li, F. Y.; Liu, C.; Huang, C. H. Synthesis, characterization, and in vivo targeted imaging of amine-functionalized rare-earth up-converting nanophosphors. Biomaterials 2009, 30, 5592−5600. (48) Zhou, J.; Sun, Y.; Du, X. X.; Xiong, L. Q.; Hu, H.; Li, F. Y. Dualmodality in vivo imaging using rare-earth nanocrystals with nearinfrared to near-infrared (NIR-to-NIR) upconversion luminescence and magnetic resonance properties. Biomaterials 2010, 31, 3287−3295. (49) Iversen, T. G.; Skotland, T.; Sandvig, K. Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nano Today 2011, 6, 176−185.

1182

dx.doi.org/10.1021/la500131d | Langmuir 2014, 30, 1176−1182