A Dual-Color Luminescent Localized Drug Delivery System with

Article pubs.acs.org/journal/abseba

A Dual-Color Luminescent Localized Drug Delivery System with Ratiometric-Monitored Doxorubicin Release Functionalities Yike Fu,† Xiaoyi Chen,‡ Xiaozhou Mou,‡ Zhaohui Ren,† Xiang Li,*,† and Gaorong Han*,† †

State Key Laboratory of Silicon Materials, School of Materials Science and Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, P. R. China ‡ Clinical Research Institute, Zhejiang Provincial People’s Hospital, Hangzhou, Zhejiang 310014, P. R. China S Supporting Information *

ABSTRACT: Implantable localized drug delivery systems (LDDSs) have been intensively investigated for cancer therapy. However, the anticancer agent release behavior as well as the local therapeutic process in the complex physiological environment remains a dark zone and consequently hinders their clinical applications. Herein, a series of Er3+doped electrospun strontium titanate (SrTiO3, STO) nanofibers with refined microstructural characteristics were exploited as a localized carrier for doxorubicin (DOX) delivery due to its light-responsive functionalities as well as expected biocompatibility. The highest DOX loading capacity and sustained releasing kinetics were obtained from the nanofibers with the highest surface area and lowest pore dimensions. Consequently, such nanofibers presented stronger in vitro anticancer efficacy to Hep G2 cells compared to that of other samples. More importantly, the amount of drug released was monitored by the ratio of green-to-red emission (I550/I660) due to the fluorescence resonance energy transfer (FRET) effect built between DOX molecules and upconversion photoluminescent nanofibers. The selective quenching effect of green emission due to DOX molecules was gradually weakened with drug releasing progress, whereas the intensity of red emission barely changed, resulting in an increased I550/I660 ratio. Such color evolution can be feasibly visualized by the naked eye. Monitoring with a spectral intensity ratio eliminates the disturbance of uncertainties in the complex physiological environment compared to just referring to the emission intensity. Such dual-color luminescent STO:Er nanofibers, designed based on the FRET mechanism, are therefore considered to be a promising new LDDS platform with ratiometric-monitored DOX release functionalities for future localized tumor therapeutic strategies. KEYWORDS: doxorubicin, localized drug delivery system, fluorescence resonance energy transfer (FRET), SrTiO3:Er nanofibers, electrospinning

1. INTRODUCTION Cancer is currently one of the main causes of death for mankind, and conventional chemotherapy continues to be the common therapeutic strategy. However, in nanoparticle-based systemic drug delivery (SDD) administration, anticancer drugs are transported into circulation around the entire body. In general, a maximum dose of the drug is initially induced, followed by rapid clearance over time, resulting in considerable side effects and limited therapeutic efficacy.1−3 It has been reported that only a rather low percentage (98.0%; Sinopharm Chemical Reagent Co., Ltd.), and erbium nitrate pentahydrate (Er(NO3)3·5H2O, >99.9%; Aladdin) were dissolved in 5 mL of acetic acid (C2H4O2, 99.5%; Sinopharm Chemical Reagent Co., Ltd.) to prepare solution B with different Er3+ concentrations (1, 2, 3, 4, and 5 mol %). Subsequently, solution B was added to solution A dropwise and stirred for 4 h to maintain the homogeneous solution. For STO:Er nanofibers with different microstructural characteristics (STO:M0, STO:M1, STO-M2) to be obtained, three types of surfactant configurations were used in solution A, as listed in Table 1.

Table 1. Surfactant Configurations Used for Three STO:Er Nanofibers with Controlled Microstructures fiber series

PVP (g/mL)

F127 (g/mL)

CTAB (g/mL)

STO-MO STO-M1 STO-M2

0.08 0.08 0.08

0 0.04 0.04

0 0 0.06

During electrospinning, the precursor was injected into a stainless steel needle, which is connected to a power supply (Glassman, PS/ FC30P04.0-22, USA). The flow rate was controlled at 0.4 mL/h, and the distance from needle tip to the grounded collector was maintained at 12 cm. A high speed monitor (Baumer, TXG04h, Germany) was used to examine the jetting mode. When the needle was applied with a voltage between 9 and 11 kV, the stable cone-jet electrospinning mode was achieved, and uniform precursor nanofibers were formed and collected, as demonstrated in Figure. S1. The as-spun nanofibers were pyrolyzed at 400 °C for 0.5 h and subsequently sintered at 700 °C for 2 h in air. 2.2. Loading and Release of Drug. DOX was chosen as a model anticancer drug for further investigation. All types of STO:Er nanofibers (50 mg) were mixed with DOX aqueous solution (20 mL, 1 mg/mL) at room temperature and shaken for 24 h to reach the equilibrium state. The DOX-loaded nanofibers were then centrifuged and washed to remove DOX molecules absorbed at the surface with deionized water. The in vitro DOX-releasing examination was operated by adding the DOX-loaded nanofibers to phosphate buffered saline (PBS, 20 mL, pH 7.4) solution under gentle shaking at 37 ± 0.1 °C. At predetermined time intervals, 5 mL of buffer solution was collected and immediately replaced with fresh PBS of equal volume. The amounts of DOX released in the supernatant solutions were examined using a UV−vis spectrophotometer (TU-1810, China) at a maximum wavelength (λmax) of 490 nm. Three measurements were performed for each sample. 2.3. In Vitro Cytotoxicity Assay. Bone marrow-derived mesenchymal stem cells (BMSCs) were employed for in vitro cytotoxicity evaluation of STO:Er nanofibers via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In a typical procedure, BMSCs were seeded in a 96-well plate at an initial density of approximately 6000−8000 cells/well and cultured in 5% CO2 at 37 B

DOI: 10.1021/acsbiomaterials.6b00046 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 1. (a) UC emission spectra of STO:Er nanofibers with different doping levels; (b) XRD patterns of SrTiO3 and 3 mol % Er-doped SrTiO3 nanofibers. (inset) Enlarged XRD patterns.

Figure 2. SEM and TEM images of STO:Er nanofibers with different surfactants: (a) as-spun STO-M0, (b) as-spun STO-M1, (c) as-spun STO-M2, (d) calcined STO-M0, (e) calcined STO-M1, and (f) calcined STO-M2. (insets) Diameter distributions of STO:Er nanofibers. °C for 24 h. The STO:Er nanofibers were disinfected with alcohol for 2 days, and then a series of nanofibers with different concentrations were incubated with cells for 1, 3, and 5 days in 5% CO2 at 37 °C. The concentrations of nanofibers were set at 0, 100, 200, 500, and 1000 μg/mL, respectively. At the end of each incubation time interval, MTT (20 μL, 10 mg/mL) solution was added to each well, and the cells were cultured for 4 h at 37 °C. The STO:Er nanofiber-containing medium was then removed and replaced with 150 μL of dimethyl sulfoxide (DMSO) per well. The absorbance was monitored via a microplate reader (Thermo Scientific Inc., USA) at the wavelength of 490 nm. The measurement was repeated six times to ensure accuracy. 2.4. In Vitro Anticancer Efficacy Assay. Similarly, serial dilutions of DOX-loaded STO:Er nanofibers were cultured with hepatoma cells (Hep G2) for 12, 24, and 48 h to evaluate the in vitro anticancer efficacy. Four concentrations at 25, 50, 100, and 200 μg/mL of unloaded nanofibers were selected to compare the influence of drug loading capacity and releasing kinetics on the inhibition effect to cancer cells. All DOX-loaded STO:Er nanofibers were immersed in PBS solution for 48 h before cell culture. The subsequent procedures of cell culture were maintained in the same was as in the cytotoxicity assay above. 2.5. Characterization. The morphology of STO:Er nanofibers was investigated via field-emission scanning electron microscopy (FESEM, Hitachi SU-70; Japan) and high-resolution transmission electron microscopy (HRTEM, Tecnai F20; FEI, USA). The phase structure was measured via an X-ray diffraction instrument (XRD, X’Pert PRO MPD; The Netherlands) operating at 40 mA and 40 kV using Cu Kα radiation. The scanning range was set between 10° and 90° with a 0.167° step size. N2 adsorption/desorption analysis was performed to

examine the surface textural characteristics of nanofibers in liquid nitrogen (77 K) via a coulter OMNISORP-100 apparatus. A PerkinElmer 580B infrared spectrophotometer (Tensor 27, Bruker, Germany) was used to record the FTIR spectra on KBr pellets. Thermogravimetry/differential scanning calorimetry (TG-DSC, DSCQ1000; TA Instruments, USA) was performed with a heating rate of 10 °C/min from room temperature to 1000 °C in air to investigate calcination conditions and DOX drug loading capacity. The upconversion photoluminescence spectra were measured under continuous 980 nm excitation from a fluorescence spectrophotometer (PL, FLSP920, Edinburgh) at room temperature. For experimental uncertainties to be minimized, the positions of samples and the spectra collection were maintained in identical conditions. Fifty fibers were chosen stochastically from SEM images and measured to obtain the diameter distribution. The nanofiber textural characteristics were calculated from five samples to obtain the average values. 2.6. Statistical Analysis. Data were collected from six replicates for each experiment. Statistical significance was assessed by Student’s t test. The level of significance was set at p < 0.05 (95% confidence level).

3. RESULTS AND DISCUSSION 3.1. Synthesis of STO:Er LDDS. The TG-DSC curves of as-spun nanofibers are shown in Figure S2 to simulate the calcination process in air at 10 °C/min with approximately 14, 40, 3.5, and 5.5% weight losses present at 64.93, 307.53, 450.15, and 560.12 °C, respectively. This is due to the removal of the remaining volatile solvent, the decomposition of inorganic salts C


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ACS Biomaterials Science & Engineering and PVP, the formation of SrCO3, and the transition from SrCO3 to perovskite SrTiO3. No apparent weight loss occurs above 700 °C, demonstrating the formation of stable perovskite SrTiO3 phase. Therefore, calcination at 700 °C for 2 h was determined as the ultimate postheating condition. A variety of STO:Er nanofibers with doping concentrations varying from 1 to 5 mol % were synthesized under the same conditions. All prepared nanofibers possess typical upconversion photoluminescence spectra under excitation of 980 nm spectrum (Figure 1a). The UC PL spectra of STO:Er nanofibers consist of green (530 and 550 nm) and red (660 nm) emissions. The green emission peaks are induced by the (2H11/2, 4S3/2) → 4I15/2 transition, and the red emission is attributed to the 4F9/2 → 4 I15/2 transition, which is similar to what other studies have reported.40,41 The emission intensity is quenched when the doping concentration is below or exceeds 3 mol %. Meanwhile, the Er doping concentration does not have an apparent influence on the fiber morphology or diameter (Figure S3). Therefore, 3 mol %, which induces the highest luminescence intensity, was chosen as the optimal Er doping concentration. The X-ray diffraction patterns of pure SrTiO3 and 3 mol % Erdoped SrTiO3 nanofibers confirm that both nanofibers possess characteristic diffraction peaks of the cubic SrTiO3 phase without any impurity phases (Figure 1b). The enlarged (110) peak of STO:Er shifts to a higher angle as shown in the the inset. In addition, the unit cell parameters of STO and STO:Er calculated from XRD patterns are a = b = c = 3.9185 Å and a = b = c = 3.8189 Å, respectively. The findings above indicate shrinkage of the crystal lattice. The Shannon effective ionic radii of Er3+ (89 pm VI, 100.4 pm VIII) is of lower dimension compared to Sr2+ (118 pm VI) at site A and larger than Ti4+ (60.5 pm VI).42 The shrinkage of the crystal lattice results from the replacement of Sr2+ with Er3+ atoms. It is therefore confirmed that Er atoms have been successfully doped into the SrTiO3 crystal lattice at Sr2+ sites. The morphology of STO:Er nanofibers prepared with various surfactant recipes was characterized by SEM and TEM. As shown in Figure 2, all as-spun STO:Er nanofibers before calcination present smooth surface morphology and similar dimensions (280−320 nm). The insets indicate nanofiber diameter distributions. After calcination at 700 °C for 2 h, the surface of nanofibers roughens markedly, and the fiber diameter is reduced to 220−260 nm, as induced by the organic surfactant decomposition and inorganic salts and the densification of STO:Er crystallites in the fibers. The internal microstructure characteristics of nanofibers were characterized further via TEM. As shown in the insets of Figure 2d−f, highly porous features of all nanofibers can be visualized because of the different electron penetrability of various zones of the nanofibers. The porous structure is enhanced in STO-M0, STO-M1, and STO-M2 in turn. The d-spacings of crystallites measured from HRTEM images are approximately 0.277, 0.384, and 0.388 nm, which are assigned to the (110), (100), and (100) planes of SrTiO3, respectively (Figure S4). The spotty diffraction patterns indicate high crystallinity of all three STO:Er nanofibers. The findings agree well with the results of XRD examination. For the effects of surfactants on fiber microstructures to be determined in a more quantitative manner, N2 adsorption/ desorption analysis was carried out to examine the surface area and porosity of STO:Er nanofibers. All samples show representative IV-type isotherms, indicating the porous microstructure of nanofibers (Figure 3). The isotherms reveal

Figure 3. N2 adsorption/desorption isotherm of STO:Er nanofibers with different surfactant configurations.

remarkable differences in the pore dimensions and surface area of STO:Er nanofibers with the increased surfactants. As summarized in Table 2, when PVP is the only surfactant, the Table 2. Textural Properties of STO:Er Nanofibers with Different Surfactant Configurations fiber series

BET surface area (m2/g)

pore volume (cm3/g)

pore size (nm)

STO-MO STO-M1 STO-M2

14.6 20.6 26.3

0.072 0.074 0.076

20.5 13.5 10.3

surface area is 14.6 m2/g, and the mean pore size is ∼20 nm. STO-M1 nanofibers show an increased surface area (20.6 m2/ g) and decreased pore size (∼14 nm). The largest surface area of 26.3 m2/g as well as smallest pore size of ∼10 nm is present in STO-M2 fibers. Furthermore, it is found that the different surfactant configurations do not induce distinguishable changes in pore volume. The as-spun STO:Er nanofibers are amorphous, containing large PVP molecules and self-assembled micelles of F127 and CTAB molecules. The pore structures form with the elimination of these surfactants during the calcination process. The varied porous structure of nanofibers is attributed to the different dimensions of micelles formed in the precursor fibers. Adding F127 and CTAB molecules, which have smaller sizes than PVP molecules, induce the formation of pores with much lower dimensions, and thus the mean pore dimension on the fibers is decreased from STO-M0 to STOM2 in turn. Meanwhile, the surface area increases because of the increased pore numbers, as demonstrated in Figure S5. Such effective manipulation of surface area and pore size via surfactant configuration control during the fabrication may facilitate tunable drug loading and releasing properties. Cytotoxicity is always a crucial factor when a biomaterial is expected to be functionalized as a localized drug delivery system. STO:Er nanofibers were cultured with BMSCs for MTT assays to assess cytotoxicity. The 1, 3, and 5 day viabilities of BMSCs cultured with STO:Er nanofibers at different concentrations (0.1−1 mg/mL) were studied. The cell proliferation exhibits an increasing trend in an incubation time-dependent manner from the lowest concentration of 0.1 mg/mL to the highest of 1 mg/mL, confirming that the STO:Er nanofibers synthesized are of satisfactory cytocompatibility (Figure 4). For each time interval, no significant differences were observed between samples with varied concentrations. One notable fact is that the nanofibers with 1 mg/mL concentration have induced a slightly lower cell D


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The DOX loading capacities of different STO:Er nanofibers were investigated via TG analysis (Figure 5b). The weight loss of pure STO:Er nanofibers is ∼2%, which results from the removal of adsorbed water. The most dramatic weight loss of DOX molecules occurs at approximately 210 and 470 °C (Figure S7). All DOX-loaded STO:Er nanofibers exhibit apparent weight loss at these two temperature points, as expected. With the increment of surfactants, the weight loss of DOX-loaded STO:Er nanofibers increases from approximately 2.5 to 9.5 wt %. The main weight loss resulted from the decomposition of DOX drug loaded on the nanofibers. Therefore, the loading capacities of STO-M0, STO-M1, and STO-M2 nanofibers are approximately 2.5, 6.3, and 9.5 wt %, respectively. The remarkably promoted drug loading capacity is ascribed to the increased surface area of STO:Er nanofibers. The upconversion emission spectra of nanofibers before and after DOX loading under 980 nm NIR excitation were further examined. As shown in Figure 6, before drug loading, all three systems with different surfactant configurations exhibit similar upconversion emissions and stronger green emission (2H11/2, 4 S3/2 → 4I15/2 transition) than red emission (4F9/2 → 4I15/2 transition). The optical image inserted in Figure 6a, captured using a digital camera, presents the typical UC luminescent phenomenon of STO:Er nanofibers under 980 nm NIR excitation, illustrating the dominated green emission of unloaded samples. The intensity ratio of red emission to green emission (I660/I550) is 0.51, 0.52, and 0.57 for the STOM0, STO-M1, and STO-M2 fibers, respectively. More importantly, the absorption spectrum of DOX was measured by a UV−vis spectrophotometer. A splendid spectral overlap is observed between the absorption curve of DOX drug and the upconversion emission of STO:Er nanofibers (Figure 6a). When nanofibers are loaded with DOX molecules, the upconversion emission changes dramatically. As shown in Figure 6b, the red emission governs the spectra for all types of STO:Er nanofibers, which is entirely opposite to that of the unloaded nanofibers. In addition, the intensity ratio of red-togreen emission exhibits a significant ascending characteristic from 13.03 to 15.35 to 18.66 with the varied surfactant configurations (from STO-M0, STO-M1, to STO-M2, respectively) (Figure 6c). In contrast, the ratio of unloaded nanofibers remains at a similar level. The mechanism is mainly attributed to the overlap between the absorption spectrum of DOX and the upconversion emission of nanofibers. The presence of a FRET effect from green upconversion photoluminescence to the absorption of DOX quenches the green emission. With the increased DOX loading capacity from STO-

Figure 4. Viabilities of BMSCs incubated with STO:Er nanofibers at different concentrations for 1, 3, and 5 days; *p < 0.05.

viability after being cultured for 5 days. Such marginally suppressed viability could be attributed to the inevitable ion release caused by the long-term unchanged culture medium. Chemical stability is a vital matter for LDDS as it relates to possible biotoxicity caused by decomposition of materials. A series of STO-M2 nanofibers were immersed in PBS solution at two different pH values (7.4 and 4.7) for different periods of time. All nanofibers exhibit similar diameters and microstructural characteristics before and after immersion in PBS solutions with different pH values (Figure S6). No sign of fiber decomposition can be observed. The STO:Er nanofibers synthesized exhibit sound chemical stability in the simulated physiological environment. 3.2. Drug Loading. The identification of chemical groups at the surface of materials was examined. The FTIR spectra of STO:Er nanofibers, pure DOX drug, and DOX-loaded STO:Er nanofibers are illustrated in Figure 5a. For unloaded nanofibers, a feature peak at 566 cm−1 is assigned to Ti−O bands. After loading with DOX, typical absorption bands assigned to the stretching vibration of CO at 1618 and 1581 cm−1 from the stretching vibration of carbonyl groups located at the anthraquinone ring of DOX molecules are observed.43 In addition, the characteristic bands at 1724 and 1280 cm−1 are ascribed to the stretching vibration of carbonyl groups at the 13-keto position and the skeleton vibration of DOX molecules, respectively.44,45 The FTIR spectrum of DOX-loaded STO:Er nanofibers combines the characteristic peaks of pure DOX molecules and STO:Er nanofibers, indicating the successful loading procedure of the DOX drug.

Figure 5. (a) FTIR spectra of STO:Er nanofibers, DOX-loaded STO:Er nanofibers, and pure DOX molecules; (b) TG curves of STO:Er nanofibers and three DOX-loaded STO:Er nanofibers. E


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Figure 6. (a) UC emission spectra before DOX drug loading; (b) UC emission spectra after DOX drug loading; and (c) intensity ratio of red-togreen emission of three STO:Er systems before and after DOX drug loading. (insets) Fluorescence photographs of nanofibers excited by 980 nm laser (a) before and (b) after DOX loading.

surfactants, the DOX-loaded nanofibers present more sustained release kinetics. The STO-M2 nanofibers, fabricated with PVP, F127, and CTAB, show the tardiest releasing behavior. Only less than 30% of DOX drug is released into PBS solution within the first 20 h, and ∼45% of the total DOX amount is liberated after a longer 140 h period. The release of DOX loaded in the STO-M1 system presents a moderate rate between those of STO-M0 and STO-M2. Thus, it is verified that the STO-M2 system does not only possess the highest DOX drug loading capacity but also induces the most lasting release manner. This is ascribed to the controlled microstructure characteristics of STO-M2 nanofibers, such as improved surface area and reduced pore dimensions. Therefore, the DOX loading and releasing behaviors of STO:Er nanofibers have been manipulated successfully. 3.4. In Vitro Anticancer Efficacy Assay. To further evaluate the impact of controlled DOX release kinetics to the in vitro anticancer efficacy of DOX-loaded STO:Er nanofibers, all nanofibers were immersed in PBS solution for 48 h prior to cell culture. As shown in Figure 8, all DOX-loaded STO:Er nanofibers present inhibition effect to the proliferation of tumor cells at all time points, indicating that the STO:Er nanofibers was pharmacologically active as an anticancer agent nanocarrier, as expected. In addition, the viability of Hep G2 cells on STOM2 was slightly lower than STO-M0 and STO-M1 nanofibers at each time point. As demonstrated in the cumulative drug release profiles of three DOX-loaded STO:Er nanofibers, the residual DOX dosage in STO-M2 after 48 h releasing is much higher than the other two due to its higher drug storage and sustained release kinetics, inducing high level of inhibition zone and in vitro anticancer efficacy for a prolonged period. One notable fact is that, under the circumstance that the similar drug content is reached and maintained at the cancerous region, the nanoparticle-based drug delivery system may have stronger efficacy, comparing to the nanofiber-based localized

M0 to STO-M2, a steady decline in green emission (500−560 nm) is observed (inset of Figure 6b), which is attributed to the improved energy transfer from green glowing nanofibers to DOX.14 In contrast, there is no obvious overlap between the DOX absorption spectrum and red upconversion emission, inducing the barely changed intensity of red upconversion emission from 630 to 730 nm, as expected.9 Therefore, the intensity ratio changes after drug loading in different systems with varied drug loading capacity. 3.3. Drug Release. Three types of DOX-loaded STO:Er nanofibers were immersed in PBS to investigate the amount of drug released as a function of immersion time. DOX molecules were entrapped within the pore structures by impregnation initially and released into the buffer saline following a diffusioncontrolled mechanism.46,47 As shown in Figure 7, all systems exhibit comparatively sustained releasing properties. DOX molecules release more swiftly in the STO-M0 system than in the other two. In the first 20 h, ∼40% of the total DOX load is released, and ∼75% is liberated in 140 h. With the increased

Figure 7. Cumulative drug release profiles of three DOX-loaded STO:Er nanofibers. F


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Figure 8. Proliferation viability histograms of Hep G2 cells cultured on DOX-loaded STO:Er nanofibers with unloaded nanofiber concentrations at (a) 25 μg/mL, (b) 50 μg/mL, (c) 100 μg/mL and (d) 200 μg/mL.

Figure 9. UC emission spectra and enlarged spectra from 500 to 600 nm as a function of release time for DOX-loaded (a, c) STO-M0 and (b, d) STO-M2 nanofibers.

been reported recently to confirm such opinion.48−50 The STO:Er nanofiber LDDS has thus been considered as a promising alternative for thermotherapy. One needed to be clarified is that, for the tumors which are hard to be located or has metastasized, particulate drug delivery may offer significantly higher efficacy for highly morbid systemic therapy. 3.5. Photoluminescence Evolution during DOX Releasing. The photoluminescence phenomenon of STO:Er nanofibers has shown a well correspondence to the DOX releasing progress. The intensity of upconversion green

drug delivery system, due to its effective cellular up-taking effect. However, comparing to the well-documented particulate drug delivery systems, the nanofiber LDDS is implanted directly to the cancerous zone, and in consequence the major drug content loaded is released locally at the tumor site while avoiding the excessive drug circulation. Therefore, although the nanofiber LDDS is out of cells when functioning, the therapeutic efficacy is enhanced, to some extent, due to such accurate delivery manner, restricting the current side effects. Many therapeutic protocols using fiber-based system have also G


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Figure 10. FTIR spectra and enlarged characteristic peaks of DOX-loaded STO-M2 nanofibers at different release times.

Figure 11. (a) I550/I660 of DOX-loaded nanofibers as a function of release time; linear correlation between I550/I660 and the percentage of DOX released with (b) STO-M0 and (c) STO-M2 nanofibers.

the drug release process. The scattering efficiency of red emission is lower than that of green, and therefore, the apparent yellowish glow can be observed by the naked eye even when a low percentage of green emission is mixed with the red emission. Nevertheless, the ratio rises in a much tardier manner in STO-M2 from 0.05 to 0.08, which is in agreement with the variation trend of green and red emission in Figure 9. The increasing rate of I550/I660 corresponds well to the DOX releasing behaviors of these two systems. Rapid DOX release kinetics induces a fast I550/I660 ratio increase and vice versa. In addition, a linear relationship between I550/I660 and the percentage of DOX released were investigated (Figure 11b,c). For STO-M0 fibers, the I550/I660 ratio (y axis) shows a linear relationship with the amount of DOX molecules (x axis) released in the measured range of 0 < x < ∼80%. The correlation equation is I550/I660 = 0.0011 DOX released (%) + 0.07 (correlation coefficient R2 = 0.95). Similarly, a well-fitted linear relationship also exists in the STO-M2 system. The correlation equation is I550/I660 = 0.0007 DOX released (%) + 0.05 (correlation coefficient R2 = 0.98). The findings above reveal that the I550/I660 signal of STO:Er nanofibers can be utilized as a high-performance reporter to quantitatively detect the amount of DOX drug released through the FRET effect.

emission between 500 to 660 nm gradually recovers along with DOX release progress, while the fluctuation of red emission around ∼660 nm is almost negligible (Figure 9). Further, the recovery rate of green emission in STO-M0 system is more rapid than STO-M2 system, which is attributed to the fast DOX releasing rate in STO-M0 system. The mechanism of such phenomenon was analyzed via FTIR examination. The peaks from 1550 to 1700 cm−1, corresponding to the stretching vibration of carbonyl groups located at the anthraquinone ring of DOX molecules, gradually weaken during drug liberation over time (Figure 10). The release of DOX molecules into PBS increases the donor-to-acceptor separation distance. Generally, FRET does not occur when the distance from donors to acceptors is greater than 10 nm.51 The DOX molecules liberated from the pores of STO:Er nanofibers lead to the increase of distance between DOX acceptor and the nanofiber donor (>10 nm), which eventually weakens the FRET phenomenon. The findings indicate that upconversion photoluminescence signals are so sensitive that it can be utilized as an ideal probe for drug release monitoring. The relationship between the intensity ratio of green-to-red emission (I550/I660) and DOX release time was analyzed in detail to demonstrate the in vitro ratiometric monitoring functionalities for DOX release behavior from STO:Er nanofibers. As shown in Figure 11a, the I550/I660 ratio increases along with the drug release for all systems as expected. It increases from 0.077 to 0.16 as DOX is released within a period of 144 h in the STO-M0 system. The insets indicate that the color of STO:Er nanofibers changes from red to yellowish over

4. CONCLUSIONS A series of electrospun STO:Er nanofibers with manipulated microstructural characteristics were successfully achieved via the surfactant control. When three surfactants (PVP, F127, and CTAB) were combined for the electrospinning precursor, H


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STO:Er nanofibers present the highest surface area and lowest pore dimensions (∼10 nm). Consequently, the higher DOX loading capacity, more sustained release kinetics, as well as stronger in vitro anticancer efficacy were achieved when using STO-M2 nanofibers as compared to those of the other two samples. More importantly, because of the construction of a FRET effect between DOX molecules and STO:Er nanofibers, the intensity ratio of green to red emission, I550/I660 ratio, corresponds effectively with DOX-releasing progress. STO:Er nanofibers (STO-M2) with the most sustained DOX release kinetics induce the tardiest increase of the I550/I660 ratio and vice versa. Furthermore, a linear correlation between the I550/ I660 ratio and the percentage of DOX molecules released was uncovered, and this can be utilized as a standard curve for evaluating the DOX releasing kinetics in a quantitative manner. The photoluminescence intensity ratio, utilized as a probe, excludes the uncertain local concentration effect compared to monitoring based only on spectral emission intensity. Such new STO:Er nanofibers with ratiometrically monitored DOX release functionalities, designed based on the FRET mechanism, have therefore provided a robust and promising platform for modern localized tumor therapeutic strategies.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00046. Schematic diagram of electrospinning, TG-DSC curve of STO:Er nanofibers,SEM images of SrTiO3 nanofibers with different Er doping concentrations, TEM and HRTEM patterns of nanofibers, schematic diagram for the formation of three STO:Er nanofibers, SEM images of STO-M2 nanofibers immersed in PBS with different pH levels for various periods of time, and TG curve of the STO:Er nanofibers and pure DOX molecule (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Nature Science Foundation of China (No. 51232006, 81301326) and the Nature Science Foundation of Zhejiang Province (LY15E020005).

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ABBREVIATIONS STO:Er, Er -doped strontium titanate; UC, upconversion; PL, photoluminescence; BMSCs, bone marrow-derived mesenchymal stem cells

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REFERENCES

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A Dual-Color Luminescent Localized Drug Delivery System with

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