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A Protein−Polymer Bioconjugate-Coated Upconversion Nanosystem for Simultaneous Tumor Cell Imaging, Photodynamic Therapy, and Chemotherapy Chunhong Dong,†,∥ Zhongyun Liu,‡,∥ Sheng Wang,† Bin Zheng,† Weisheng Guo,† Weitao Yang,† Xiaoqun Gong,† Xiaoli Wu,† Hanjie Wang,*,† and Jin Chang*,† †

School of Life Sciences, School of Materials Science and Engineering, Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment Technology, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin 300072, P. R. China ‡ Key Laboratory of Coastal Environmental Processes and Ecological Remediation, Research Center for Coastal Environmental Engineering and Technology of Shandong Province, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai City, Shandong Province 264003, P. R. China S Supporting Information *

ABSTRACT: Combined cancer therapy possesses many advantages including improved tumoricidal efficacy, reduced side effects, and retarded drug resistance. Herein, a protein−polymer bioconjugate-coated multifunctional upconversion nanosystem, consisting of upconversion nanoparticles (UCNs) core, tailored amphiphilic protein−polymer bioconjugate shell, and photosensitizer zinc phthalocyanine (ZnPc) and antitumor drug doxorubicin coloaded inside, was elaborately developed for combined photodynamic therapy (PDT) and chemotherapy. In this system, UCNs core could convert deep penetrating nearinfrared light to visible light for simultaneous cell fluorescence imaging and photodynamic therapy by activating ZnPc to generate cytotoxic ROS, while the protective shell of bovine serum albumin−poly(ε-caprolactone) (BSA-PCL) offered excellent water solubility, good stability, and low cytotoxicity. The ROS production test showed that this nanosystem could successfully generate singlet oxygen under NIR irradiation. A cellular uptake study demonstrated that intense fluorescence emission of the UCNs could be observed in HeLa cells, indicating their outstanding real-time imaging capability. More importantly, compared with single PDT or chemotherapy systems, the constructed combined therapy UCNs system demonstrated significantly enhanced tumor cell killing efficiency. On the basis of our findings, this multifunctional UCNs nanosystem could be a promising versatile theranostic nanoplatform for image-guided combined cancer therapy. KEYWORDS: upconversion nanosystems, protein−polymer bioconjugate, combined therapy, chemotherapy, photodynamic therapy

1. INTRODUCTION

to eliminate tumors owing to the high-dose drug required,

Cancer, as one of the major causes of mortality and morbidity globally, leads to many deaths every year.1,2 How to eliminate and prevent cancer remains a long-standing huge challenge in current medical science. Chemotherapy, one of the most wellestablished cancer treatments, has many drawbacks such as severe side effects and limited efficacy.3,4 Despite great advances and breakthroughs made in improving drug efficacy by developing nanomaterial-based drug delivery systems,5−7 a single chemotherapy system is still not as effective as expected

metastasis, and development of drug resistance.8−10 Currently,

© XXXX American Chemical Society

combined therapy, an integration of two or more therapeutic forms, is becoming increasingly popular in modern treatment technology of cancer with the superior advantages of synergistic Received: September 17, 2016 Accepted: November 16, 2016 Published: November 16, 2016 A

DOI: 10.1021/acsami.6b11803 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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PDT therapy systems, significantly enhanced tumor cell killing efficiency was readily realized by the DOX-loaded UCN/ ZnPc@BSA-PCL nanosystem via effective cooperation of photodynamic therapy and chemotherapy. Taken together, multiple exciting advantages including uniform size, good water solubility, low cytotoxicity, real-time fluorescence imaging, NIR-mediated PDT, and enhanced cancer therapy have been well integrated in this system. The BSA-PCL bioconjugatebased multifunctional UCN nanosystem displayed great potential for future real-time image-guided high-efficiency tumor therapy.

anticancer effects, minimized toxic side effects, maximized therapeutic outcomes, and suppressed drug resistance.11−14 Photodynamic therapy (PDT), a mild, noninvasive, and minimally toxic therapeutic strategy based on photochemical reactions of photosensitizers (PSs) to generate cytotoxic reactive oxygen species (ROS) to kill cells, has emerged as an attractive and pioneering tumor therapeutic modality due to improved selectivity and remote light controllability.15,16 Combined photodynamic/chemotherapy has achieved remarkably improved cancer therapeutic efficacy than respective monotherapies.17−19 Especially in recent years, near-infrared light-mediated upconversion nanoparticles (UCNs)-based PDT systems with the capacity to address the problems of photodamage and limited tissue penetration depth of conventional PDT systems have attracted considerable interest and intense attention.20−22 Unfortunately, however, high-performance UCNs, available photosensitizers, and anticancer drugs are generally hydrophobic.23−25 Therefore, further surface engineering and modification become an urgent and necessary issue. To addresses this problem, various strategies have been developed, including ligand exchange, silica coating, amphiphilic polymer modification, and so on.26−30 Thereinto, amphiphilic polymer modification becomes one of the most prevailing strategies for its convenience, flexibility, stability, and versatility.31−33 Among all the amphiphilic polymers, a type of newly emerging biomaterial, tadpole-like amphiphilic protein− polymer conjugate, is becoming more and more popular in the field of nanomedicine for its superior performance and versatile functions.34−36 The hydrophilic protein segment as the head of the tadpole offers excellent biocompatibility, low immunogenicity, low nonspecific protein adsorption, and inherent biofunctional properties, while the polymer component as its tail imparts amphiphilic self-assembly property, diversity, good stability, and some other fascinating properties.37,38 Recently, we have reported a biodegradable protein−polymer bioconjugate with bovine serum albumin (BSA) and poly(εcaprolactone) (PCL) by controllable maleimide−thiol reactions.39 The content and conjugation site of attached PCL and further self-assembly behaviors of the conjugates be well controlled in a precise and reproducible way. As a kind of amphiphilic biohybrid, BSA-PCL has been successfully employed for targeted delivery of doxorubicin (DOX) for single tumor chemotherapy in our previous work. In our present study, we further introduced BSA-PCL for UCNs surface modification for the first time and developed a combined therapy system of DOX-loaded UCN/ZnPc@FABSA-PCL for simultaneous tumor cell imaging and enhanced combined photodynamic therapy and chemotherapy. The system was fabricated by co-encapsulation of UCNs, photosensitizer, and DOX into the hydrophobic core of the selfassembled nanostructures of BSA-PCL via a straightforward and robust ultrasonication-induced assembly method. In this system, the outer BSA protection shell could endow it with great water solubility, high biocompatibility, and good stability in physiological environments, while PCL segment and inherent hydrophobic region of BSA could contribute to a high loading capability of both ZnPc and DOX. Under 980 nm light irradiation, UCNs could convert deeply penetrating nearinfrared light to visible light for simultaneous real-time cell fluorescence imaging and photodynamic therapy by activating ZnPc photosensitizer to generate cytotoxic ROS. Compared with single DOX-induced chemotherapy or single UCNs-based

2. MATERIALS AND METHODS 2.1. Materials. The hydrophobic NaYF4:Yb/Er (Y/Yb/Er = 78/ 20/2) UCNs were prepared by using a well-established protocol in the mixture of 1-octadecene and oleic acid as previously reported.40 BSA (lyophilized powder), dithiothreitol (DTT), zinc phthalocyanine (ZnPc), folic acid (FA), 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA), and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich and used without further purification. BSA was used after reduced by DTT, and maleimide-functionalized polycaprolactone (Mal-PCL) was prepared via ring-opening polymerization of ε-caprolactone with stannous octoate as catalyst and N-(2hydroxyethyl)maleimide as the initiator according to the reported method.39 N-(3-(Dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were obtained from GL Biochem Ltd. (Shanghai, China). All other chemicals and solvents were of reagent grade, obtained from local suppliers, and used without any further treatment. 2.2. Characterization. To characterize the optical properties of UCNs, their UV−vis absorption spectra and photoluminescence (PL) spectra were separately measured by a Shimadzu UV-2450 spectrophotometer and fiber spectrometer with a 980 nm laser. The pure UCNs and obtained UCNs nanosystems were visualized by a Technai G2 20-STWIN transmission electron microscope (TEM) in the bright-field mode. Dilute suspensions of pure UCNs in chloroform or UCNs nanosystems in water were dropped onto a carbon-coated copper grid and then air-dried at room temperature prior to examination under TEM. Fourier transform infrared (FTIR) spectra of the UCNs before and after the assembly of amphiphilic BSA-PCL molecules were measured by a Spectrum 65 FT-IR spectrometer. The particle size and zeta potential of the prepared UCNs nanosystems were measured in PBS buffer (pH 7.4) by dynamic light scattering (DLS) using a Zetasizer Nano-ZS equipped with a 633 nm He−Ne laser from Malvern Instruments dynamic light scattering (DLS) at 25 °C. As for cell experiments, the HeLa cells were observed by an Olympus fluorescence microscope. 2.3. Synthesis of BSA-PCL and FA-BSA-PCL Bioconjugate. The protein−polymer bioconjugate BSA-PCL was synthesized by coupling Mal-PCL with a molecular weight of 3500 Da to reduced BSA as we previously reported.39 FA-BSA-PCL was synthesized based on carbodiimine chemistry with EDC and NHS as the cross-linker. In brief, FA (5 mg), NHS (2 mg), and EDC·HCl (3 mg) were dissolved in 2 mL of dimethyl sulfoxide (DMSO) and stirred at 0 °C for 2 h. Then BSA-PCL (50 mg) dissolved in 20 mL of PBS (0.01 M, pH 7.4) was added. The reaction mixture was kept at room temperature under vigorous stirring overnight. After dialyzed in distilled water (MWCO = 8000) for 2 days, the solution was lyophilized under vacuum to obtain dry products. 2.4. Preparation of UCN@BSA-PCL. In this work, to examine the modification performance of BSA-PCL on hydrophobic UCNs, UCN@BSA-PCL was first fabricated via an ultrasonication-induced method as previously reported.41,42 A typical preparation process was as follows: (i) 6−7 mg of BSA-PCL was dissolved in 3 mL of deionized water in a small beaker. (ii) UCNs were precipitated by ethanol and dissolved in dichloromethane to a final concentration of 20.0 mg/mL. (iii) Under the pulsed ultrasonication (every 3 s for a B


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ACS Applied Materials & Interfaces duration of 3 s at 200 W), 100 μL of UCNs solution was injected dropwise via a syringe into the BSA-PCL solution in a cold−water bath. Then the solution was kept for 10 min to form an emulsus solution. (iv) The emulsus solution was then evaporated to remove dichloromethane until completely clear. (v) Residual polymers were removed by centrifugation at 15000g for 20 min, and the obtained UCN@BSA-PCL precipitate was washed with double-distilled water for three times and finally kept at 4 °C for further use. Herein, an ultrasonic cell crushing instrument (JY92-IID, Ningbo Scientz Biotechnology Co., LTD) was employed. 2.5. Assembly of DOX-Loaded UCN@BSA-PCL, UCN/ZnPc@ BSA-PCL, and DOX-Loaded UCN/ZnPc@BSA-PCL. The assembly of DOX-loaded UCN@BSA-PCL, UCN/ZnPc@BSA-PCL, and DOXloaded UCN/ZnPc@BSA-PCL was similar to the above-mentioned fabrication process of UCN@BSA-PCL based on the simple, fast, and robust ultrasonication-induced assembly method. Take for example the case of DOX-loaded UCN/ZnPc@BSA-PCL. In brief, a mixture solution of 40 μL of UCNs solution (20 mg/mL in dichloromethane), 100 μL of ZnPc solution (4 mg/mL in THF), and 100 μL of DOX solution (10 mg/mL in dichloromethane) was injected dropwise via a syringe into the 3−4 mL of BSA-PCL solution (2 mg/mL in doubledistilled water) in a cold−water bath under the pulsed ultrasonication. In the case of folic acid (FA)-decorated systems, the process was similar, except that BSA-PCL was replaced by FA-BSA-PCL. 2.6. Determination of Drug Loading Efficiency. The drug loading efficiency was determined based on the UV−vis spectrophotometry by using a Shimadzu UV-2450 spectrophotometer. The loading efficiencies of ZnPc and DOX loaded in the UCNs nanosystems were calculated by the absorbance at their characteristic absorbance peak at 674 and 490 nm after subtracting the corresponsive absorbance contribution from UCNs@BSA-PCL before drug loading at the same nanoparticle concentration. Briefly, the standard curves of absorbance vs concentration were made by separately measuring the absorbance of pure ZnPc at 674 nm and DOX at 490 nm at five different concentrations. Then a certain amount of prepared UCNs nanosystems was diluted to a known concentration (mass: mNanosystem), and its absorbance was measured and used to obtain the mass of ZnPc or DOX (mZnPc or mDOX). The drug loading efficiency of ZnPc and DOX was thus reported as the mZnPc/ mNanosystem and mDOX/mNanosystem. 2.7. In Vitro Drug Release. The in vitro release behavior of DOX and the stability of ZnPc were studied by a dialysis technique in PBS buffer solution (pH 7.4) at 37 °C in a shaking water bath. To avoid any possible mutual interference between DOX and ZnPc, we prepared DOX-loaded UCN@BSA-PCL and UCN/ZnPc@BSA-PCL separately as research samples. As DOX and ZnPc have their own colors of red and blue, the separate photos of dialysis solution at different time points for DOX-loaded UCN@BSA-PCL and UCN/ ZnPc@BSA-PCL were taken by a digital camera. Quantitive analysis was performed by UV−vis spectrophotometry using a Shimadzu UV2450 spectrophotometer. Take the release behavior of DOX, for example; the process was as follows. In brief, a solution of the DOX-loaded UCN@BSA-PCL was sealed in a dialysis bag (MWCO of 8000−14 000) and immersed in 20 mL of PBS solution (pH 7.4) under constant shaking. At the predetermined time points, 10 mL of PBS medium was taken out to measure the UV absorbance of released DOX at 490 nm and replaced by an equal volume of fresh PBS. The cumulative release amount of DOX was calculated according to the measured absorbance. Similarly, the same method was used to study the leakage behavior of ZnPc from UCN/ZnPc@BSA-PCL to assess the stability of photosensitizer within the nanocarriers. The amount of leaking ZnPc was measured according to the measured absorbance at 674 nm. 2.8. Size Stability of DOX-Loaded UCN/ZnPc@BSA-PCL. The size stability of DOX-loaded UCN/ZnPc@BSA-PCL in protein contained biofluids is quite critical for its biological applications. In this work, to further evaluate its size stability, the prepared DOXloaded UCN/ZnPc@BSA-PCL solution was gently dispersed in PBS buffer supplemented with 10% FBS (0.1 M, pH 7.4, 10% FBS) and DMEM, respectively. The particle size was monitored by dynamic light

scattering (DLS) at different incubation times. Every sample was measured at least three times and averaged. 2.9. Fluorescence Stability in Different pH Buffer Solution. The fluorescence stability of UCNs is a prerequisite for the PDT treatment. To investigate their fluorescence stability, the UCN/ ZnPc@BSA-PCL nanocarriers were incubated in PBS buffer at different pH values of 6.5, 7.4, and 8.5, respectively. The corresponding fluorescence emission spectra excited by a 980 nm laser were measured from 400 to 750 nm at different time points. The relative intensity was calculated. 2.10. Determination of Singlet Oxygen. The generation of singlet oxygen is a vital prerequisite for the application of PDT systems. ABDA, an established singlet oxygen quencher, was used in our present study as a probe to determine the generation efficiency of singlet oxygen. The generation of 1O2 would lead to the photobleaching of ABDA. In general, different experimental samples solutions were mixed with 1 mM ABDA and then irradiated by a 980 nm laser after different incubation periods of time. From the decline of the fluorescence intensity of ABDA at 433 nm, the generation of singlet oxygen (1O2) could be reflected. 2.11. Cell Studies. 2.11.1. Cell Culture. The human cervical carcinoma cell line HeLa was used to perform cell experiments including cytotoxicity, cellular uptake, and intracellular studies. HeLa cells were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin−streptomycin antibiotics, maintained at 37 °C in a humidified atmosphere with 5% CO2, and split 1:3 three times a week. For all experiments, the HeLa cells without any treatment served as negative control groups. 2.11.2. Cytotoxicity Assays. The tetrazolium-based standard 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to evaluate the potential cytotoxicity of different samples in HeLa cells. In this work, the cytotoxicity of the protein− polymer conjugate BSA-PCL, UCN@BSA-PCL, and UCN/ZnPc@ BSA-PCL was investigated. HeLa cells were seeded in a 96-well plate at a density of 5000 cells/well in DMEM and incubated for 24 h. The prepared samples of different concentrations ranging from 12.5 to 400 μg/mL in DMEM were then added into each well to replace the original medium. Following further incubation for 24 h, 20 μL of MTT stock solution (5 mg mL−1 in PBS) was added per well and maintained for an additional 4 h at 37 °C. The medium was carefully abandoned from per well, followed by adding 100 μL of DMSO. After shaking the plate for 10 min, ultraviolet absorbance values of MTT were measured by using a microplate reader at 570 nm. All the samples were tested in triplicate. The cell viability was calculated relative to the nontreated control cells. 2.11.3. Cell Uptake in Vitro. The cell uptake into HeLa cells of DOX-loaded UCN/ZnPc@BSA-PCL and DOX-loaded UCN/ZnPc@ FA-BSA-PCL was investigated at the same concentration by fluorescence microscope. HeLa cells were seeded in a 24-well plate overnight. About 50 μL of each experimental samples was respectively added to the culture medium. After incubation for predetermined time, the culture medium was removed, and the HeLa cells were washed three times with PBS buffer solution. For cell imaging study, the cells were further fixed with 4% paraformaldehyde for 15 min at 37 °C and labeled with 300 μL of DAPI solution (500 ng/mL) for 20 min for nucleus staining. At last, the cells were washed twice with PBS buffer, maintained in pure PBS, and observed by a fluorescence microscope. 2.11.4. Combined Photodynamic Therapy and Chemotherapy Studies in Vitro. After determining the singlet oxygen generation efficiency of the PDT-based nanosystem, its PDT treatment effect was further evaluated in HeLa cells. The single PDT treatment, single chemotherapy, and combined PDT−chemotherapy treatment were investigated and compared. HeLa cells were seeded in a 24-well plate overnight for cell attachment, and different samples including free DOX, DOX@BSA-PCL, UCN/ZnPc@BSA-PCL, DOX-loaded UCN/ ZnPc@BSA-PCL, and DOX-loaded UCN/ZnPc@FA-BSA-PCL were then added into respective wells. The HeLa cells were cultured for a total of 24 h at 37 °C. The standard MTT assay was used to calculate C


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Figure 1. Schematic illustration of the protein−polymer bioconjugate-based multifunctional UCN nanosystem for image-guided photodynamic therapy and chemotherapy.

Figure 2. (A) Fabrication of DOX-loaded UCN/ZnPc@FA-BSA-PCL nanosystem via an ultrasonication-based assembly method. (B) Photos of UCN@BSA-PCL (B1), DOX-loaded UCN@BSA-PCL (B2), UCN/ZnPc@BSA-PCL (B3), DOX-loaded UCN/ZnPc@BSA-PCL (B4), and DOXloaded UCN/ZnPc@FA-BSA-PCL (B5) nanocarrier before and after oil evaporation. the cell viabilities relative to the untreated control cells. As for the UCNs-based PDT experimental groups, the cells were irradiated by 980 nm laser at a power density of 1.0 W cm−2 for 10 min (2 min interval for every 2 min of light exposure to avoid heating) after incubation with nanoparticles for 6 h, followed by further incubation. Besides, the influence of 980 nm laser irradiation alone on viability of HeLa cells was evaluated. In this work, control experiments were carried out under the same cases, except the variable parameters. As for all the DOX-contained groups (free DOX, DOX-loaded UCN@BSAPCL, and DOX-loaded UCN/ZnPc@BSA-PCL), the content of DOX was kept at the same concentration of 5 μg mL−1. Meanwhile, the content of ZnPc in all the ZnPc-contained groups was also kept constant at the same concentration.

the photosensitizer to generate ROS. The combined PDT and chemotherapy lead to enhanced therapy efficiency. 3.1. Preparation, Characterization, and Optimization of UCN-Based Nanosystems. As a newly emerging type of biohybrid, protein−polymer conjugate demonstrates excellent performance in terms of water solubility, biocompatibility, assembly behavior, stability, and nonspecific protein adsorption.39 In particular, the BSA-PCL conjugate made by attaching PCL to BSA displays good biodegradability, showing great potential in the field of nanomedicine. The related characterization and self-assembly behavior of BSA-PCL have been reported in our previous work.39 In this study, in order to construct the UCN-based nanosystems, a facile and robust ultrasonication-based assembly method was employed. A schematic of the preparation process of multifunctional DOX-loaded UCN/ZnPc@FA-BSA-PCL is shown in Figure 2A. Under ultrasonication in oil−water biphase, the hydrophobic segment of amphiphilic BSA-PCL interacted with each other via hydrophobic interactions, forming a uniform protein layer around UCNs and meanwhile encapsulating hydrophobic DOX into intermediate hydro-

3. RESULTS AND DISCUSSION The performance of the protein−polymer bioconjugate-based multifunctional UCN nanosystem is illustrated in Figure 1. This nanosystem could passively accumulate in the tumor region via the enhanced permeation and retention (EPR) effect. Under 980 nm light irradiation, UCNs convert the deeply penetrating near-infrared light to visible light for simultaneous cell fluorescence imaging and photodynamic therapy by activating D


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Figure 3. (A−D) Fluorescence spectra, UV−vis absorption spectra, particle size, and photos of UCN/ZnPc@BSA-PCL with different loading amounts of ZnPc. (E) UV−vis absorption spectra of BSA-PCL, UCNs, DOX, and DOX-loaded UCN@BSA-PCL. (F) UV−vis absorption spectra of FA, BSA-PCL, and FA-BSA-PCL.

at 1654 cm−1 and amide II at 1538 cm−1. The spectrum of assembled UCNs@BSA-PCL showed both characteristic bands of BSA-PCL conjugate and characteristic bands of UCNs, indicating the successful coating of this protein−polymer conjugate on the surface of UCNs. Moreover, their micromorphologies were further characterized by TEM (Figure S5). The results indicated that all the UCN nanocarriers were monodistributed and nanoscale uniform spheres with single upconversion nanoparticle encapsulated inside. As for UCN-based PDT systems, the incorporated photosensitizer ZnPc could effectively absorb the emission light of NaYF4:Yb,Er, transfer the absorbed photon energy to nearby oxygen molecules, and thus generate cytotoxic reactive oxygen species (ROS) to kill cancer cells. Considering that the loading capacity of photosensitizer ZnPc plays a critical role in the photodynamic treatment efficiency, hereby we mainly studied the influence of added volumes of ZnPc stock solution during the assembly procedure in order to optimize the PDT system. The amounts of BSA-PCL conjugates and UCNs were kept constant. As shown in the UV−vis absorption spectrum of Figure S6, ZnPc possessed an intense absorption peak around 660 nm appeared. The ZnPc loading efficiency of UCN/ ZnPc@BSA-PCL with different added volume of ZnPc stock solution was determined by UV−vis spectrophotometry. As the

phobic layer. The self-assembled nanomicelles of BSA-PCL in this way are demonstrated in Figure S1. In this work, NaYF4:Yb,Er (Y:Yb:Er = 78:20:2) nanoparticles and zinc phthalocyanine (ZnPc) were separately used as UCNs and photosensitizer. Energy dispersive spectroscopy (EDS) spectrum and X-ray diffraction (XRD) spectrum of UCNs are shown in Figures S2 and S3, respectively. Herein, UCN@BSAPCL, DOX-loaded UCN@BSA-PCL, UCN/ZnPc@BSA-PCL, DOX-loaded UCN/ZnPc@BSA-PCL, and DOX-loaded UCN/ ZnPc@FA-BSA-PCL nanocarriers were prepared separately. From Figure 2B, we could see that emulsion-like systems were formed after ultrasonication. After further oil evaporation, all the systems changed into homogeneous and transparent carrier solutions without any precipitation, which indicated that all the BSA-PCL bioconjugate-based UCNs nanocarriers displayed great solubility and dispersibility in water due to the successful and complete assembly progress. To further confirm the assembly of amphiphilic BSA-PCL conjugates on the surface of UCNs, FT-IR analysis was performed. As shown in Figure S4, peaks at 2927 and 2857 cm−1 appearing in the spectrum of pure UCNs could be ascribed to the asymmetric (νas) and symmetric (νs) stretching vibrations of methylene (CH2) in the long alkyl chain of oleic acid ligands, respectively, and the FTIR spectrum of BSA-PCL conjugate showed the characteristic amide I peaks E


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Figure 4. TEM image of pure hydrophobic NaYF4:Yb,Er UCNs (A) and DOX-loaded UCN/ZnPc@FA-BSA-PCL (B). DLS result (C) and zeta potential (D) of DOX-loaded UCN/ZnPc@FA-BSA-PCL.

shows the UV−vis absorption spectra of BSA-PCL, FA, and FA-decorated FA-BSA-PCL. The red-shift of the absorption peak was caused by the attachment of FA. Under optimized condition mentioned above, we finally constructed the combined therapy system of DOX-loaded UCN/ZnPc@FABSA-PCL nanocarriers. The morphology, size surface chemistry, and optical properties of DOX-loaded UCN/ZnPc@FA-BSA-PCL nanocarrier were characterized by TEM, dynamic light scattering, zeta potential, optical fiber spectrometer, and UV−vis spectrophotometer. As shown in Figure 4A, the original pure UCNs were approximately 30 nm in uniform size. After the modification, the constructed DOX-loaded UCN/ZnPc@FABSA-PCL nanocarriers were also well-dispersed nanospheres with a single UCN as the core, and no aggregation occurred (Figure 4B). Their hydrodynamic size exhibited in Figure 4C was about 45 nm, a little larger than that shown in TEM image, which was probably due to the swelling effect of surface hydrophilic polymer layer in aqueous phase.43 Besides, they possessed a surface zeta potential of −10 mV. The fluorescence emission and UV−vis absorption spectra of DOX-loaded UCN/ZnPc@FA-BSA-PCL are displayed in Figures S8 and S9. Under NIR 980 nm light irradiation, the nanocarriers displayed fluorescence emission at around 550 and 660 nm, while both ZnPc and DOX showed no fluorescence. Moreover, the size stability of the DOX-loaded UCN/ZnPc@BSA-PCL nanosystem was further evaluated. As shown in Figure S10, whether incubated in PBS buffer supplemented with 10% FBS or in DMEM, the particle size of the DOX-loaded UCN/ ZnPc@BSA-PCL nanosystem changed little during the measured 48 h. Therefore, this UCNs nanosystem exhibited excellent size stability in protein contained biofluids, which was probably due to the great stability of the supermolecular assembly structures of BSA-PCL amphiphiles and the extremely low nonspecific adsorption of outer BSA layer.

added volume of ZnPc stock solution increased from 30 to 100 μL, the ZnPc loading efficiency increased from 1.59% to 5.37% (Figure S7). As shown in Figures 3A and 3B, as the ZnPc loading efficiency increased, the fluorescence emission intensity of UCN/ZnPc@BSA-PCL at around 660 nm of UCNs decreased gradually while an increasingly enhanced absorption around 660−680 nm appeared in the UV−vis absorption spectra, indicating an effective photon energy transfer from UCNs to ZnPc. Meanwhile, the hydrodynamic size of the obtained UCN/ZnPc@BSA-PCL nanocarriers varied a little under the same assembly condition, and they had a hydrodynamic size of about 44.9 nm in the case of 5.11% ZnPc loading efficiency (Figure 3C). The bright field and dark field photos of the UCN/ZnPc@BSA-PCL nanocarriers in water solution were demonstrated in Figure 3D from which a gradual color change of the fluorescence was easily observed. Generally, a larger loading amount of photosensitizer was in favor of higher photodynamic treatment efficiency. To take this into consideration, 70 μL of ZnPc stock solution was used in our later work. As for the anticancer drug DOX, the adding dosage was determined according to our earlier experience.39 The evaluated loading ratio of DOX determined by UV−vis spectrophotometry was about 7% (w/w). The ultraviolet− visible absorption spectrum of DOX-loaded UCN@BSA-PCL in Figure 3E demonstrated obvious characteristic absorption peak of DOX around 490 nm. 3.2. Preparation and Characterization of DOX-Loaded UCN/ZnPc@FA-BSA-PCL. To construct the multifunctional nanosystem combining photodynamic therapy and chemotherapy, DOX and ZnPc were simultaneously coloaded inside during the self-assembly process of BSA-PCL bioconjugates. In addition, in order to enhance tumor cell targeting ability, folic acid was further attached to BSA-PCL via carbodiimide chemistry to prepare FA-BSA-PCL for further use. Figure 3F F


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Figure 5. Fluorescence stability of UCN/ZnPc@BSA-PCL nanocarrier in PBS buffer at different values within 10 h: the fluorescence emission spectrum (left) and relative intensity in PBS buffer at pH 6.5 (A), PBS buffer at pH 7.4 (B), and PBS buffer at pH 8.5 (C).

existing hydroxyl ions, leading to their fluorescent quenching.44 Therefore, UCN/ZnPc@BSA-PCL was relative stable when stored in neutral and slightly alkaline buffers instead of acid condition. 3.4. Determination of Singlet Oxygen Production. The production of singlet oxygen is a vital prerequisite for photodynamic therapy. In our present study, an established singlet oxygen quencher, ABDA was employed to assess the generation of singlet oxygen (1O2) by UCN/ZnPc@BSA-PCL under a 980 nm laser irradiation. With the generation of 1O2 increased, the fluorescence emission intensity of ABDA decreased. Three other groups including UCN/ZnPc@BSAPCL without irradiation, UCN@BSA-PCL with 980 nm laser irradiation, and pure ZnPc with 980 nm laser irradiation were used as control groups. The relative fluorescence emission intensity change of ABDA over 980 nm laser irradiation time is shown in Figure 6. We could find that there was little change in ABDA fluorescence intensity in three control groups, indicating almost no singlet oxygen was produced. By contrast, a gradually decreased fluorescence intensity of ABDA could be obviously

To investigate the release behavior of DOX and the stability of ZnPc inside the UCN nanosystems, a dialysis technique was used. As shown in Figure S11A, the release of DOX could be easily observed during the dialysis process, especially within the first 12 h. Further quantitive analysis by UV−vis spectrophotometry in Figure S11B shows the gradual release of DOX molecules from the DOX-loaded UCN@BSA−PCL nanosystems. The cumulative release amount of DOX increased fast at the beginning, reaching around 65% within the first 12 h, and then slowed down and finally reached a plateau of 70% after 48 h. Under the same conditions, however, no visible ZnPc release could be observed for the dialysis solutions of UCN/ZnPc@ BSA-PCL were all clear and colorless (Figure S11A). Quantitive analysis by UV−vis spectrophotometry also showed that almost no ZnPc released from the UCN/ZnPc@BSA-PCL nanosystems (data not shown). The release studies demonstrated that photosensitizer ZnPc had a quite good stability inside the UCN/ZnPc@BSA-PCL probably due to the strong hydrophobic interactions and extremely poor hydrophilicity. 3.3. Fluorescence Stability in Different pH Buffer Solution. As for fluorescent nanoparticles, the fluorescence stability in biological fluids is of vital importance for their biological applications. The fluorescence stability of UCNs is critical for their photodynamic therapy efficiency and imaging performance. Herein, UCN/ZnPc@BSA-PCL nanocarriers were separately incubated in PBS buffer at different pH values of 6.5, 7.4, and 8.5, and their fluorescence emission spectra under a 980 nm laser irradiation were measured at different time points. The relative intensity was further calculated. As shown in Figure 5A, the fluorescence intensity of UCNs at 543 nm declined by 25% after storage in PBS at pH 6.5 for 8 h. By contrast, when incubated in PBS buffers at pH 7.4 and 8.5, the fluorescence emission of UCNs was relatively stable, and more than 85% of original fluorescence could be well retained after 8 h (Figure 5B,C), suggesting that the surface coating of BSA-PCL is relatively compact to protect UCNs against water environment. This result was in agreement with our previous report. When incubated at a low pH value, UCNs could be eroded by the

Figure 6. Production of singlet oxygen (1O2) of UCN-based nanocarriers under 980 nm laser irradiation displayed by the decay of ABDA fluorescence. G


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maintained quite good safety. Therefore, it had great potential in the field of constructing highly biocompatible multifunctional nanoplatforms to advance the development of nanomedicine. 3.6. Cell Uptake in Vitro. HeLa cells were used herein as model cancer cells to investigate the cell uptake performance of the multifunctional therapy system DOX-loaded UCN/ZnPc@ FA-BSA-PCL, and DOX-loaded UCN/ZnPc@BSA-PCL was employed as control to evaluate the influence of FA. To explore the change of cell uptake over incubation time, the HeLa cells separately treated with DOX-loaded UCN/ZnPc@FA-BSAPCL for 0.5, 2, and 4 h were fixed with paraformaldehyde, stained with DAPI, and finally imaged under the fluorescent microscope. To observe the fluorescence of UCNs, a 980 nm laser was used. DAPI was excited by the ultraviolet light and DOX was excited by the green light. As shown in Figure 8, as the incubation time prolonged, the fluorescence intensities of both UCNs and DOX increased gradually, suggesting that more DOX-loaded UCN/ZnPc@FABSA-PCL nanoparticles were successfully endocytosed into HeLa cells. A bright fluorescence of UCNs and DOX could be observed in cells which were incubated with DOX-loaded UCN/ZnPc@FA-BSA-PCL for 4 h. By contrast, under the same incubation time of 4 h, the cell uptake of FA-free DOXloaded UCN/ZnPc@BSA-PCL was weaker. So the attachment of FA could enhance the cell uptake of the nanoparticles by the active targeting effect. The results demonstrated that the FAdecorated multifunctional DOX-loaded UCN/ZnPc@BSAPCL could effectively be uptake into tumor cells. The UCNbased system was supposed to work for photodynamic therapy, while the loaded DOX for chemotherapy. Meanwhile, the fluorescence of UCNs could also act as imaging signals, achieving real-time image guidance in cancer therapy. 3.7. Combined Photodynamic Therapy and Chemotherapy Studies in Vitro. The enhanced chemotherapy efficiency of the combined DOX-loaded UCN/ZnPc@BSAPCL therapy system was further investigated by the standard MTT assay. Hereby, DOX@BSA-PCL for single chemotherapy and UCN/ZnPc@BSA-PCL for single PDT were used as control. Figure S10 shows the influence of NIR 980 light on the cell viability of the HeLa cells. It could be observed that the

observed when incubated with UCN/ZnPc@BSA-PCL nanocarrier under a 980 nm laser irradiation, reflecting the successful generation of singlet oxygen. Therefore, UCN/ZnPc@BSAPCL nanocarrier could be successfully used for the NIRinduced photodynamic therapy. As for the UCN-based PDT system, three key components including UCNs, ZnPc, and 980 nm light irradiation were all necessary and essential. The lack of any one of them would lead to treatment failure. 3.5. Cytotoxicity Assays. To evaluate the potential cytotoxicity of the BSA-PCL bioconjugate and our constructed UCN@BSA-PCL and UCN/ZnPc@BSA-PCL, the viability of HeLa cells treated with different concentrations of samples for 24 h was measured by the standard MTT assay. As shown in Figure 7, the cell survivals decreased slightly as the sample

Figure 7. In vitro cytotoxicity of BSA-PCL, UCN@BSA-PCL, and UCN/ZnPc@BSA-PCL measured by standard MTT assay in HeLa cells. The pure DMEM served as the control. Error bars indicate the standard deviation of three experiments.

concentration gradually increased from 12.5 to 400 μg/mL. Overall, the HeLa cell survivals treated with the three samples were all above 80% even at a relatively high concentration (400 μg/mL), indicating their low cytotoxicity against HeLa cells. In particular, the BSA-PCL bioconjugate demonstrated no obvious cytotoxicity. As a kind of amphiphilic protein−polymer biohybrid derived from BSA and PCL, the bioconjugate

Figure 8. Fluorescence images of HeLa cells treated with FA-decorated DOX-loaded UCN/ZnPc@FA-BSA-PCL for 0.5, 2, and 4 h and FA-free DOX-loaded UCN/ZnPc@BSA-PCL for 4 h. Insets are images with higher magnification times. Scale bars represent 50 μm. H


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displayed higher cell uptake in HeLa cells than nontargeted ones. More importantly, compared with single DOX-induced chemotherapy and single UCNs-based PDT therapy, significantly enhanced tumor cell killing efficiency was realized by the combined therapy nanosystem via the effective cooperation of photodynamic therapy and chemotherapy. In view of all the advantages mentioned above, this multifunctional BSA-PCL bioconjugate-based UCN nanosystem could therefore serve as a promising versatile nanoplatform for simultaneous imaging capability and high-efficiency combined tumor therapy.

irradiation of 980 nm light alone did not cause significant cell death. Besides, there was no obvious inhibition effect when the cell was treated with UCN/ZnPc@BSA-PCL without 980 nm light irradiation (Figure 9), which agreed with the MTT result mentioned above. NIR light irradiation was a prerequisite to trigger UCNs-based PDT.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b11803. TEM image of self-assembled nanomicelles of BSA-PCL bioconjugate (Figure S1); energy dispersive spectroscopy (EDS) spectrum (Figure S2) and X-ray diffraction (XRD) spectrum (Figure S3) of NaYF4:Yb,Er; FTIR spectra of pure UCNs, BSA-PCL, and the assembled UCNs@BSA-PCL (Figure S4); TEM images of UCN/ ZnPc@BSA-PCL, DOX-loaded UCN@ BSA-PCL, and DOX-loaded UCN/ZnPc@BSA-PCL nanocarriers (Figure S5); ultraviolet−visible absorption spectrum of ZnPc (Figure S6); ZnPc loading efficiency of UCN/ZnPc@ BSA-PCL with different volume of added ZnPc (Figure S7); fluorescence spectra of DOX, ZnPc, and DOXloaded UCN@BSA-PCL (Figure S8); UV−vis absorption spectrum of DOX-loaded UCN@BSA-PCL (Figure S9); particle size stability of DOX-loaded UCN/ZnPc@ FA-BSA-PCL (Figure S10); (A) photos of dialysis PBS buffer solution (pH 7.4) for DOX-loaded UCN@BSAPCL and UCN/ZnPc@BSA-PCL separately, and (B) DOX release behavior of DOX-loaded UCN@BSA-PCL in PBS buffer solution (pH 7.4) at 37 °C (Figure S11); effect of near-infrared 980 nm laser irradiation alone on HeLa cell viability (Figure S12) (PDF)

Figure 9. Proliferation inhibition effect of different samples including free DOX, DOX-loaded UCN@BSA-PCL, UCN/ZnPc@BSA-PCL, UCN/ZnPc@BSA-PCL with 980 nm laser irradiation, DOX-loaded UCN/ZnPc@BSA-PCL with 980 nm laser irradiation, and folatedecorated DOX-loaded UCN/ZnPc@FA-BSA-PCL with 980 nm laser irradiation on HeLa cells by the standard MTT assay. The data are shown as mean ± SD. Error bars are based on at least quadruplicate measurements. P values: *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed Student’s t test).

In addition, the single chemotherapy by DOX@BSA-PCL and single PDT treatment by UCN/ZnPc@BSA-PCL displayed inhibition rate of 37.6% and 37.9%, respectively. By contrast, the combined DOX-loaded UCN/ZnPc@FA-BSAPCL displayed significantly enhanced treatment efficiency, and the cell killing efficiency was about 72.4%. This enhanced treatment efficiency was probably attributed to the cooperative effect of DOX-induced chemotherapy and UCN-based PDT. Furthermore, FA-decorated DOX-loaded UCN/ZnPc@FABSA-PCL demonstrated slightly higher treatment efficiency than FA-free ones, which may be attributed to the enhanced cell uptake mentioned above. Taken together, enhanced chemotherapy efficiency was successfully achieved by the multifunctional DOX-loaded UCN/ZnPc@BSA-PCL therapy system. The combined therapy nanoplatform showed great potential for high-efficiency tumor therapy.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (J.C.). *E-mail [email protected] (H.W.). ORCID

Chunhong Dong: 0000-0001-6950-6120 Author Contributions ∥

C.D. and Z.L. have contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the National Natural Science Foundation of China (51373117, 51573128, 51303126), Key Project of Tianjin Natural Science Foundation (13JCZDJC33200, 15JCQNJC03100), National High Technology Program of China (2012AA022603), and Doctoral Base Foundation of Educational Ministry of China (20120032110027) for financial support.

4. CONCLUSIONS In summary, we reported hereby a multifunctional BSA-PCL bioconjugate-based upconversion nanosystem which simultaneously allowed imaging and provided a high efficiency combined photodynamic therapy and chemotherapy. The system with a uniform size of 45 nm demonstrated great solubility, good size and fluorescence stability, effective ROS production under 980 nm light irradiation, and low cytotoxicity. It has been demonstrated in cell uptake study that good imaging capability was achieved via intense fluorescence emission of UCNs. Besides, folic acid-decorated nanosystems

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