Biomaterials 35 (2014) 4146e4156

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An upconversion nanoparticle e Zinc phthalocyanine based nanophotosensitizer for photodynamic therapy Lu Xia a, b, Xianggui Kong a, *, Xiaomin Liu a, Langping Tu a, b, Youlin Zhang a, Yulei Chang a, Kai Liu a, Dezhen Shen a, Huiying Zhao c, Hong Zhang d, * a

State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033, China Graduate University of the Chinese Academy of Sciences, Beijing 100049, China c Department of Basic Medicine, Gerontology Department of First Bethune Hospital, University of Jilin, Changchun 130021, China d Van’t Hoff Institute for Molecular Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 January 2014 Accepted 26 January 2014 Available online 14 February 2014

Recent advances in NIR triggering upconversion-based photodynamic therapy have led to substantial improvements in upconversion-based nanophotosensitizers. How to obtain the high efficiency of singlet oxygen generation under low 980 nm radiation dosage still remains a challenge. A highly efficient nanophotosensitizer, denoted as UCNPs-ZnPc, was constructed for photodynamic therapy, which is based on near infrared (NIR) light upconversion nanoparticle (UCNP) and Zn(II)-phthalocyanine (ZnPc) photosensitizer (PS). The high 1O2 production efficiency came from the enhancement of the 660 nm upconversion emission of NaYF4:Yb3þ, Er3þ UCNP with 25% Yb3þ doping, covalent assemblage of UCNP and ZnPc which significantly shortened the distance and enhanced the energy transfer between the two. The high 1O2 production led to a secure and efficient PDT treatment, as evidenced by the in vivo test where UCNPs-ZnPc of 50 mg per kg body weight was locally injected into the liver tumor in mice, a low 980 nm radiation dose of 351 J/cm2 (0.39 W/cm2) and short irradiation duration of 15 min were sufficient to perform image-guided PDT and caused the liver tumor inhibitory ratio of approximately 80.1%. Histological analysis revealed no pathological changes and inflammatory response in heart, lung, kidney, liver or spleen. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Upconversion ZnPc Photodynamic therapy Covalent assembly

1. Introduction Photodynamic therapy (PDT) is a minimally invasive medical treating strategy involving photosensitizer (PS), light, and tissue oxygen at ground state oxygen of triplet (3O2) which can generate the aggressive singlet oxygen (1O2) [1e3]. As far as photosensitizers were concerned, Zinc (II)-phthalocyanine (ZnPc) have been proven highly selective for tumor targeting and showed enhanced cytotoxic effects both in vitro against several cell lines as well as in vivo in mouse tumor models [4e6]. On the other hand, current PDT in clinic is focused on superficial lesions due to the penetration depth restriction of the short wavelength excitation light [7]. Another limitation is the low veracity of the treatment because tumor tissue can be located only subjectively and approximately via a clinical endoscope, however the tumor cells cannot be accurately located [8,9]. Therefore, image-guided or visual treatment of tumor during

* Corresponding authors. E-mail address: [email protected] (X. Kong). 0142-9612/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2014.01.068

PDT is currently attracting special attention. For image-guided PDT, two lights of different wavelengths are often adopted to realize imaging and therapy, respectively, which makes it difficult for realtime monitoring and PDT efficacy assessment. Upconversion luminescence nanoparticles (UCNPs) doped with lanthanide ion (Ln3þ, such as Er3þ, Tm3þ, Ho3þ), which have multicolor upconversion emissions from ultraviolet to visible range, are highly stable and can be used for photodynamic diagnosis (PDD) and/or PDT under the excitation of 980 nm NIR light [10e13]. In particular, using UCNPs for bioimaging, i.e. PDD, can improve signal-to-noise ratio of biodetection due to the lack of autofluorescence. Moreover, UCNPs are safer for bioapplications relative to fluorescent organic dyes, quantum dots and so on [14,15], due to the lack of chemically induced free radicals and photoinduced reactive oxygen intermediates which result in nucleic acid strand breakage and nucleobase damage [16]. Therefore, UCNPs based nanophotosensitizers are considered to possess potential for image-guided PDT [17e24]. However, most reported laser power of triggering UCNPs based nanophotosensitizers for PDT is too high and the exposure time is in general too long which

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might be related with low upconversion luminescence efficiency and/or quantum yield of 1O2 [1,18,21,25,26,29]. In practice, a secure and efficient PDT requires a high quantum yield for 1O2 generation, low irradiation intensity and short irradiation time of light in a given area. 1O2 production of the UCNP-based nanophotosensitizer is dependent on the energy transfer efficiency which relies on the spectral overlap between the donor and acceptor, the distance between the two, and the amount of PS molecules loaded on each UCNP. Up to now, various UCNPs based nanophotosensitizers have been reported for PDT [25e31]. The most popular strategy of loading PS to UCNPs is to graft PS in porous silica shell or polymers via physical adsorption or physical encapsulation [1,25,32]. The advantage of this strategy is their simplicity and relative high loading capacity. But it is obvious that efficiency of FRET between UCNP and PS is greatly affected because the SiO2 shell increases the distance between UCNPs and PS. In addition, when physically entrapped inside the silica network or the hydrophobic layer, those PS molecules may be prematurely released from the UCNPs which can lead to a reduced efficiency of treatment. Covalently coupling of PS molecules onto the surface of UCNPs may be able to overcome the drawback [33]. In order to enhance the efficiency of FRET between UCNPs and PS, and thus obtain the high quantum yield of 1 O2, we have reported a covalent bonding strategy to link rose bengal (RB) onto UCNPs, which had a high 1O2 production enabling direct detection of the fluorescence of 1O2 at 1.27 mm, however, relatively large 980 nm radiation dosage of 900 J/cm2 was still necessary to have an observable therapeutic effect [18]. To meet the demand of high 1O2 production yield, in this work, we plan to covalently assemble the nanophotosensitizers (UCNPsZnPc/FA) of UCNPs, Zinc (II) phthalocyanine and folic acid, which can be triggered by 980 nm light irradiation. Upconversion luminescence at 540 nm will be used for PDD, whereas the branch at 660 nm to trigger ZnPc for PDT (Scheme 1). To guarantee a high FRET efficiency between UCNPs and PS, which is essential for high quantum yield of 1O2, we shall improve the upconversion emission spectrum of Er3þ ions to such that its 660 nm band is much stronger than that of 540 nm via increasing the doping concentration of Yb3þ ions. The resulted high 1O2 production shall lead to a secure and efficient image-guided PDT treatment. In addition, histological analysis shall be carried out to reveal pathological changes and inflammatory response in heart, lung, kidney, liver and spleen after PDT treatment with intratumoral direct injection of UCNPs-ZnPc/FA nanophotosensitizers. 2. Materials and methods

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Zinc (II)-phthalocyanine (ZnPc), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxy-succinimide, 1,3-diphenylisobenzofuran (DPBF), PEG succinimidyl carbonate (PEG-SC), MTT, folic acid (FA), DAPI, paraformaldehyde were purchased from SigmaeAldrich. All chemicals were used as received without further purification. 2.2. Synthesis of amino-functionalized NaYF4:Yb3þ, Er3þ UCNPs

b-NaYF4:Yb (20% or 25%, respectively), Er (2%) nanocrystals was synthesized following a literature procedure reported [32]. The particles were resuspended in cyclohexane. In order to obtain amino-functionalized upconversion nanoparticles, a ligand exchange approach was adopted using Poly(allylamine) (PAAm) to transform the hydrophobic NaYF4:Yb3þ, Er3þ nanoparticles into hydrophilic ones. 50 ml PAAm (20 wt%) aqueous solution were added to 4 ml ethanol to form a clear solution after being dispersed with ultrasonic wave. Then 0.2 ml of 17 mmol/ml solution of NaYF4:Yb3þ, Er3þ nanocrystals in cyclohexane were added. The solution was stirred at room temperature for 36 h. The nanoparticles were obtained after centrifugation (11,000 rpm, 4  C, 30 min) and redispersed in water. In the resulting system, PAAm has replaced Oleic acid as a ligand. The PAAm coated UCNPs provide a terminal amino which can be used for covalently UCNPs with carboxyl terminated molecules. 2.3. Covalent conjugation of NaYF4:Yb3þ, Er3þ UCNPs with ZnPc photosensitizers 2, 9, 16, 23-tetracarboxylic Zinc phthalocyanine were synthesized according to literature methods [34]. To covalently conjugate 2, 9, 16, 23-tetracarboxylic Zinc phthalocyanine to amino-functionalized NaYF4:Yb3þ, Er3þ UCNPs, 5 mL of DMF solution, containing different amount of 2, 9, 16, 23-tetracarboxylic Zinc phthalocyanine (0.2 mg, 0.4 mg, 0.8 mg, 1.6 mg, 3.2 mg), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (0.2 mg, 0.4 mg, 0.8 mg, 1.6 mg, 3.2 mg), and N-hydroxy-succinimide (0.2 mg, 0.4 mg, 0.8 mg, 1.6 mg, 3.2 mg),were incubated at room temperature for 2 h, and then 10 mg of amino-functionalized NaYF4:Yb3þ, Er3þ UCNPs was added into the solution and stirred vigorously for 24 h. UCNPs-ZnPc conjugates were then centrifuged and washed with DMF to remove any unreacted 2, 9, 16, 23tetracarboxylic Zinc phthalocyanine. The amount of photosensitizer attached to UCNPs was calculated from the 2, 9, 16, 23-tetracarboxylic Zinc phthalocyanine absorption spectrum. To study further the stability of the covalently bonded UCNPsZnPc conjugates, different amount of added Zinc phthalocyanine (2 mg/ml) (50 ml, 100 ml, 200 ml, 400 ml, 800 ml, 1600 ml) was also linked with 10 mg of UCNPs via electrostatic interaction and followed by the same washing procedure with DMF. Both conjugates formed via electrostatic and covalent bonding were then washed by DMF, in which 2, 9, 16, 23-tetracarboxylic Zinc phthalocyanine and Zinc phthalocyanine can dissolve well, followed by centrifugal separation. The process was repeated three times. The eluate was characterized by UV/vis absorption after each time separation. Using the eluate instead of the conjugates to study the stability was based on the fact that scattering of the conjugates was severe, which makes quantitative comparison difficult. 2.4. Singlet oxygen detection A chemical probe DPBF is used to confirm singlet oxygen by detection its absorption intensity at 410 nm via UVeVis spectroscopy [35]. In a typical DPBF experiment, 20 ml ethanol solution of DPBF (10 mmol/L) was added to 2 ml of a UCNPs-ZnPc solution and transferred into a 10 mm cuvette. The solution was kept in the dark and irradiated with a 980 nm laser for 4 min, and the absorption intensity of DPBF at 410 nm was recorded every 1 min. For the control experiments, DPBF absorption was also recorded for comparison at the same conditions in the absence of UCNPs-ZnPc or 980 nm irradiation.

2.1. Materials

2.5. Amino group detection

YCl3$6H2O (99.9%), YbCl3$6H2O (99.9%), ErCl3$6H2O (99.9%), NaOH (98%), NH4F (98%), 1-octadecene (90%), Oleic acid (OA), octadecene (ODE), Poly(allylamine) (PAAm),1,2,4-Benzenetricarboxylic anhydride, ammonium molybdat, Zinc chloride,

The existence of amino group on the surface of nanoparticles was proved by fluorometric method using non-fluorescent fluorescamine reagent for rapid amino assay [40]. The reaction of primary amines with fluorescamine can result in

Scheme 1. The construction and operating principle of the UCNPs-ZnPc nanophotosensitizer.

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fluorophore products, and the excess fluorescamine can be hydrolyzed into nonfluorescent products very fast. By measuring the emission band centered at 470 nm (lex ¼ 390 nm), the presence of amino group can be validated.

of cell lysate was recorded at 490 nm by a microtiter plate reader, and the cell viability could be calculated from the average value of four parallel wells. 2.9. Upconversion luminescent imaging of cells

2.6. Surface modification of the nanoparticles with FA and PEG To covalently conjugate 2, 9, 16, 23-tetracarboxylic Zinc phthalocyanine and folic acid on UCNPs, 5 ml of dimethylformamide solution containing 0.8 mg of 2, 9, 16, 23tetracarboxylic Zinc phthalocyanine, 0.5 mg of folic acid, 1 mg of 1-ethyl-3- (3dimethylaminopropyl) carbodiimide and 1 mg of N-hydroxy-succinimide were incubated at room temperature for 2 h, and then 10 mg of amino-functionalized NaYF4:Yb3þ, Er3þ upconversion nanoparticles was added into the solution and stirred vigorously for 24 h. The mixture was then centrifuged at 10,000 rpm for 10 min three times to spin down the nanoparticles. The supernatant was withdrawn carefully and the particles resuspended in DMSO and ethanol mixture solution (DMSO: ethanol ¼ 4:1) for further used. To reduce the undesired toxicity of nanoparticles to normal tissue, the surface of UCNPs-ZnPc/FA were coated with PEG. 4 ml of DMSO and ethanol mixture solution containing 1 mg PEG-SC and 1 mg UCNPsZnPc/FA was shaken by shaking table. The mixture was then centrifuged at 10,000 rpm for 10 min to spin down the nanoparticles. The supernatant was withdrawn carefully and the particles resuspended in PBS (PH ¼ 7.4). The washing process was repeated twice. 2.7. To assess the NIR exposure effect on the cells Hela cells that over express folate receptors and A549 that have a low expression of folate receptors were purchased from First Bethune Hospital, University of Jilin. HeLa cells were grown in a 96-well cell-culture plate at 104e105 per well and then incubated for 24 h at 37  C under 5% CO2. A power adjustable 980 nm fiber laser with maximal output power of 30 W (n-LIGHT Corporation) was collimated and employed as area light source to irradiate the 96-well plate. After 10 min exposure of 980 nm light at different power density (0, 0.39, 0.85, 1.23, 1.61, 1.99 W/cm2), the cells were allowed to incubate for an additional 48 h. To assess the effect of exposure to NIR laser on the cells, the cell viability was measured by 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. 2.8. Targeted cancer cell imaging and therapy Hela cells cultured in folate-free RPMI-1640 medium was considered as the positive group, while HeLa cells cultured in normal RPMI-1640 medium and A549 cells which were cultured in folate-free RPMI-1640 medium were considered as a negative group. All the mediums were supplemented with 10% fetal bovine serum, 100 mg/mL penicillin, and 100 mg/mL streptomycin. Cells were cultivated in medium at 37  C in a humidified 95% air and 5% carbon dioxide (CO2) atmosphere. The cytotoxicity was measured using a standard MTT assay. HeLa cells were grown in a 96-well cell-culture plate at 104e105 per well and then incubated for 24 h at 37  C under 5% CO2. After 24 h culturing, UCNPs-ZnPc/FA nanoconjugates were added to the culture medium at different concentrations, with five parallel wells for each concentration (0, 100, 200, 400, 800 mg/mL). The standard MTT assay was carried out to determine the cell viabilities relative to the untreated cells. For the FA targeted PDT experiment, the cells were allowed to incubate with different concentration UCNPs-ZnPc/FA (0, 100, 200, 400, 800 mg/mL) for another 24 h at 37  C and then washed twice with PBS before being exposed to NIR irradiation. A power adjustable 980 nm fiber laser with maximal output power of 30 W was collimated and employed as area light source to irradiate the 96-well plate. After 10 min exposure of 980 nm light at 0.39 W/cm2, the cells were allowed to incubate for an additional 48 h. To assess the effect of exposure to NIR laser on the cells with and without the nanoparticles, the cell viability was measured by MTT assay. Typically, 10 mL of MTT solution (5 mg/mL MTT in PBS) was added to each well and incubated for 4 h at 37  C. After removing the medium, the wells were washed by PBS, and then the intracellular formazan crystals were extracted into 100 ml of DMSO. The absorbance

For the FA targeted imaging experiment, both positive and negative cells were seeded in the confocal dishes at a concentration of 103/ml. After 24 h of cell attachment, both positive and negative cells were incubated with 200 mg/mL UCNPsZnPc/FA for 2 h at 37  C. Before imaging all cells were washed three times and fixed in 4% paraformaldehyde for 20 min at RT. Cells were washed twice with PBS three times. The nuclei were then counterstained with 0.1 mg/mL DAPI for 10 min at RT followed by twice washing with PBS for three times. Upconversion fluorescence imaging were then performed using a Nikon Inverted Microscope Eclipse Ti-U Main Body (Nikon, Tokyo, Japan) equipped with C2-SHS Scanner and Controller under excitation of lower power density (0.19 W/cm2). 2.10. In vivo PDT Female C57/6J mice (20 g, 6e8 weeks old) used in this study were purchased from First Bethune Hospital, University of Jilin. All experiments were carried out in compliance with the animal management. The Hepa1-6 tumor model was established by subcutaneously inoculating Hepa1-6 cells (3  106) into the upper axillary fossa in the mice (n ¼ 6). The mice were investigated when the tumor grew to a diameter of 4e6 mm. 100 ml saline or UCNPs-ZnPc/FA (10 mg/ml) was intratumorally injected into each Hepa1-6 tumor-bearing mouse. The mice were randomly assigned into four groups treated with different injections, as follows: (1) group 1 received only intratumoral injection of the saline (the control group, n ¼ 6); (2) group 2 received intratumoral injection of the saline with NIR light irradiation (n ¼ 6); (3) group 3 received intratumoral injection of the UCNPs-ZnPc/FA (n ¼ 6); (4) group 4 received intratumoral injection of the UCNPs-ZnPc/FA with NIR light irradiation (n ¼ 6). The tumors were irradiated with a 980 nm laser light (0.39 W cm2) for 15 min. To avoid any tissue damage by heating, the laser treatment was done with 3 min interval for every 3 min of light exposure. After treatment, the tumor volume was calculated as length  (width)2  1/2 with a caliper over 2 weeks. The body weight of each mouse was monitored every other day over 2 weeks. Relative tumor volume, relative body weight and inhibition ratio were defined as follows: Relative tumor volume ¼ V/V0, V0 and V stand for the tumor volume on the initial day and on the day of measurement, respectively. Relative body weight ¼ W/W0, W0 and W are the body weight of mouse on the initial day and on the day of measurement, respectively. Inhibition ratio ¼ (Vc  Vt)/Vc  100%, Vt and Vc represent the average tumor volume for the control group and treatment group, respectively. 2.11. Histology examination Histology analysis was performed at the 14th day after treatment. The organs (heart, liver, spleen, lung and kidney) and tumor tissues of the mice in the control group and treatment group were isolated from the mice, fixed with 10% neutral buffered formalin and embedded in paraffin. The sliced organs and tumor tissues (8 mm) were stained with Hematoxylin and Eosin (H&E) and examined by a microscope.

3. Results and discussion 3.1. Synthesis of UCNPs and covalently constructing UCNPs-ZnPc/FA nanophotosensitizer The NaYF4: 25% Yb3þ, 0.2% Er3þ oleic acid coated UCNPs were used as the donor, instead of 20% Yb3þ used generally in the current studies to increase the upconversion luminescence (UCL) at 660 nm (Fig. S1) and their morphologies and phase purities were analyzed by TEM and XRD, respectively, as shown in Fig. 1 and Fig. S2. Good

Fig. 1. TEM image of NaYF4:Yb3þ, Er3þ UCNPs (a) before phase transfer and (b) after phase transfer.

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Fig. 2. FTIR transmission spectra of amino-functionalized NaYF4:Yb3þ, Er3þ UCNPs (black curve), ZnPc (COOH)4 (blue curve) and UCNPs-ZnPc nanoconjugates (green curve). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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monodispersity of the UCNPs is witnessed by TEM where the average diameter is estimated about 30 nm. The diffraction peaks of these UCNPs can be indexed as a pure hexagonal phase of NaYF4. The hydrophilic NH2-functionalized UCNPs were prepared via a ligand exchanging process using PAAm as surface coating agent. The existence of amino group on the surface of UCNPs was proved by fluorometric method using non-fluorescent fluorescamine reagent for rapid amino assay (Fig. S3) [40]. To ensure that the majority of ZnPc molecules were firmly linked to UCNPs, a covalent conjugation strategy was followed that involved a crosslinking reaction between the amino group of the UCNPs and the carboxyl group of the 2, 9, 16, 23-tetracarboxylic Zinc phthalocyanine (ZnPc(COOH)4) [34]. Subsequently, PEG succinimidyl carbonate (PEG-SC), which has a good compatibility with biological systems and can reduce the undesired toxicity of UCNPs, was used to stabilize the nanocomposites in biological media. The covalent conjugation of UCNPs with ZnPc was confirmed from FTIR absorption spectra in Fig. 2. For a free ZnPc(COOH)4, the C]O stretching vibration mode and the OeH stretching vibration mode of the carboxyl group are located at 1710 cm1 and 923 cm1, respectively. After conjugating with UCNPs, the peak at 1710 cm1 disappeared and two new peaks appeared at 1660 and 1519 cm1, corresponding respectively to the C]O stretching vibration and Ne H bending vibration modes of secondary amide.

Fig. 3. (a) UVeVis absorption spectra of UCNPs nanoconjugates covalently bounded with different concentration of ZnPc(COOH)4. The weight percentage of ZnPc (COOH)4 per UCNPs nanoconjugates is also given. (b) Consumption of DPBF over time with 980 nm irradiation due to 1O2 generation from different weight percentage of ZnPc(COOH)4 per UCNPs nanoconjugates constructed in the covalent way. (c) Change of the absorption intensities of ZnPc with increasing the amount of added ZnPc non-convalently absorbed to UCPNs. The inset shows UVeVIS absorption spectra of UCNPs non-covalently absorbed with ZnPc at different added values. (d) Consumption of DPBF over time due to 1O2 generation for different weight percentage of non-convalently absorbed ZnPc with the same power density 980 nm irradiation.

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Fig. 4. Absorption spectra of eluate after washing with DMF 1, 2, and 3 times the physically adsorbed UCNPs-ZnPc complexes (solid lines), and corresponding absorption spectra of the eluate the for covalently bonded UCNPs-ZnPc/FA nanoconjugates are shown in inset (dashed lines).

The ZnPc loading capacity could be determined by characterization of the UVeVIS spectra and singlet oxygen production of UCNPs-ZnPc complexes. It can be seen from Fig. 3a that the absorption intensities remarkably raised up with the amount of added ZnPc. Fig. 3b shows the corresponding singlet oxygen production determined by the chemical probe 1, 3 diphenylisobenzofuran (DPBF) [35], from which the optimal loading capacity of ZnPc was determined to be 6% (w/w). For the sake of comparison, we also investigated the capability of ZnPc loaded with UCNPs through physical absorbed approach and its singlet oxygen production. As

shown in Fig. 3c, the absorption intensities raised up with the amount of added ZnPc. The physical absorbed ability for ZnPc only reached to w1.5% (w/w) when the quantity of ZnPc increased to over 400 ml (2 mg/ml). The corresponding UVeVIS absorption spectra of UCNPs non-covalently absorbed with ZnPc at different added values are also given in the inset. Fig. 3d shows the corresponding singlet oxygen production determined by the DPBF with 980 nm irradiation which is under the same condition of covalent UCNPs-ZnPc. It can be seen obviously that only 10% DPBF was consumed at its highest loading amount (1.5% w/w), whereas nearly 74% DPBF was depleted over 4 min at its optimal loading for the covalently assembled UCNPs-ZnPc conjugates. It demonstrated that our nanophotosensitizers constructed covalently is more effective in loading photosensitizer and producing singlet oxygen. Compared to noncovalently bonding approaches adopted generally such as electrostatic interactions, covalently bonding strategy is supposed to be very robust, which is in line with our results shown in Fig. 4, where the ZnPc eluate after each time washing with DMSO was characterized by UV/VIS absorption. Comparison between the absorption spectra of the eluate of covalently and non-covalently loading UCNPs-ZnPc, leads to the conclusion that, contrary to ZnPc desorption from electrostatically assembled UCNPs-ZnPc complexes, the amount of ZnPc in the eluate is two order of magnitude less than that of covalently bonded UCNPs-ZnPc conjugates. 3.2. Energy transfer from UCNPs to ZnPc In this study, the multifunctional UCNPs nanophotosensitizers were constructed on the basis of nonradiative energy transfer, due to the overlap of red emission at 660 nm of NaYF4:Yb3þ, Er3þ UCNPs, rather than the one at 540 nm, and 680 nm absorption of ZnPc(COOH)4 photosensitizers (Fig. 5a). According to the Förster

Fig. 5. (a) Upconversion luminescence spectrum of UCNPs (black curve) as donor and the absorption spectrum of the acceptor ZnPc(COOH)4 (blue curve) as acceptor. (b) Upconversion luminescence spectra of UCNPs (black curve) and UCNPs-ZnPc/FA conjugates (green curve) under excitation of 980 nm. Photographs of (c) UCNPs and (d) UCNPsZnPc/FA nanoconjugates under ambient light (left) and under 980 nm laser irradiation (right), respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Luminescence decay curves of upconversion emissions monitored (a) at 540 nm, (b) at 660 nm for covalently bonded UCNPs-ZnPc/FA nanoconjugates (blue), physically adsorbed UCNPs-ZnPc complexes (green) and void UCNPs (red). RB fluorescence decay was monitored at 720 nm (c). Best fitting curves are also shown as a solid line (black). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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theory [36,37], the emission intensity of red UCL band at 660 nm was 2.69 times higher than that of 540 nm. As mentioned above, ZnPc(COOH)4 is a very efficient photosensitizer in producing singlet oxygen. Comparing with our previous work based on covalently assembled UCNPs-RB nanophotosensitizers, the value of overlap integral of UCNPs and ZnPc is 1.43 times larger than that of UCNPs and RB. These results reveal that the efficiency of energy transfer is higher in UCNPs-ZnPc than that in UCNPs-RB. The energy transfer from UCNPs to ZnPc (COOH)4 is confirmed from both the steady-state UCL spectra and the decay kinetics. The UCL spectra of Fig. 5b demonstrate evidently that the 660 nm band was strongly quenched by ZnPc(COOH)4 under excitation of 980 nm, while the 540 nm band remained unchanged. This can be visualized from the obvious fluorescent color changes of the pure UCNPs and the UCNPs-PS conjugates, as shown in Fig. 5c and d, respectively. Under excitation of 980 nm CW laser, UCNPs appeared yellow (Fig. 5c, right), while UCNPs-PS nanoconjugates became green (Fig. 5d, right), indicating that the upconversion red light was completely quenched. The Förster resonance energy transfer (FRET) efficiency can be estimated from the quenching of red UCL: E ¼ (I0  I1)/I0, where I0 and I1 are red emission intensities of free UCNPs and UCNPs-PS nanoconjugates, respectively [38]. Based on this formula, the energy transfer efficiency was determined as high as 80.9% for the present covalently bonded nanoconjugates. The high energy transfer efficiency is attributed to the covalent strategy to cross-link photosensitizers and larger overlap integral of UCL at 660 nm and absorption at 680 nm of ZnPc as mentioned above, which shortened the distance between UCNPs and PS and avoided desorption of ZnPc, compared with the previous reported protocols such as the silica shell encapsulating method [1,21,30,32]. To further illustrate the efficient energy transfer in the covalent UCNPs-ZnPc complexes, non-covalent bonded UCNPs-ZnPc complexes were constructed for comparison. The temporal behaviors of UCL were shown in Fig. 6a at 540 nm and Fig. 6b at 660 nm for the UCNPs-ZnPc conjugates assembled covalently (blue) and noncovalently (green) and non-functionalized UCNPs (red) sample. In the present case, the fact that the average decay time at 660 nm decreases obviously from 368 ms to 192 ms for the covalent UCNPsZnPc conjugate, while just a slight change from 368 ms to 324 ms for the non-covalent absorption UCNPs-ZnPc conjugates, confirming the higher efficient energy transfer in our covalent model. The decay kinetics at 540 nm for both shows only slight difference (from 205 ms to 175 ms in the covalent model and from 205 ms to 186 ms in the non-covalent model). Furthermore, the fluorescence lifetime of ZnPc monitored at 720 nm is completely different between the direct excitation using laser of 680 nm and the FRET from UCL at 660 nm. The fluorescence decay time of ZnPc, which is several nanoseconds for free ZnPc, lengthened to 294 ms when adopting the FRET between ZnPc and UCNPs (Fig. 6c). This efficient FRET is the main reason for the high quantum yield of singlet oxygen in covalently assembled UCNPs-ZnPc nanoconjugates. 3.3. Singlet oxygen production from UCNPs-ZnPc nanoconjugates As shown in Fig. 7a, the absorption intensity of DPBF monitored at 410 nm decreases significantly with 980 nm irradiation time when incubated with UCNPs-PS nanoconjugates, the absorption at 680 nm is for ZnPc molecules. Fig. 7b gives the relationship between DPBF consumption and the irradiation time. The control experimental results are shown in Fig. 7b, where the upper two lines (correspond to red and black line) are for DPBF incubated with UCNPs-ZnPc nanoconjugates without 980 nm irradiation and for DPBF with 980 irradiation, respectively, and the bottom lines (green) is for DPBF incubated with UCNPs-ZnPc with 980 nm irradiation. It is clear that the DPBF consumption remains unchanged

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Fig. 7. (a) UVeVis absorption spectra of the DMF solution containing DPBF and UCNPs-ZnPc/FA after different irradiation times (0e4 min) with a 980 nm irradiation. (b) Consumption of DPBF over time (green); others were control experiments without UCNPs (black) or NIR (red), respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. (a) UCL images and specificity of the UCNPs-ZnPc/FA nanophotosensitizers under excitation of 980 nm with lower power (0.19 W/cm2). Hela cells cultured in folate-free medium (top, positive) and in folate-supplemented medium (middle, negative). The negative control is also performed with A549 cells (bottom). (b) Bright field (b(I)) and ULC (b(II)) images of Hela cells incubated with UCNPs-ZnPc/FA before PDT treatment; bright field (b(III)) and ULC (b(IV)) images of Hela cells after PDT treatment.

for the upper two, indicating a lack of singlet oxygen generation. This observation illustrates that singlet oxygen could be generated only from the cooperation of UCNPs and the ZnPc photosensitizers via an efficient energy transfer process. After 4 min irradiation nearly 74% of the DPBF was consumed which is very efficient compared with the nanophotosensitizers constructed by silica shell adsorbing, where the PS loading efficiency is relative lower and the silica shell itself might hamper the encapsulated PS from interacting with molecular oxygen dissolved in solution [39]. In this work, the strategy of the covalent conjugation of UCNPs with ZnPs, surmounted these shortages because the photosensitizer molecules were covalently bonded to the UNCP, which greatly enhances the interaction between the ZnPc and UCNPs, thus resulting in the high yield of singlet oxygen.

version) images of cells are from the UCL at 540 nm and 660 nm, respectively. It is apparent that the UCNPs-ZnPc/FA conjugations were mainly located in the cells (Fig. 8a, top), illustrating the specific targeting ability of the nanophotosensitizers. The absence of

3.4. Cancer cell imaging and photodynamic therapy To demonstrate the cancer cell targeting imaging and PDT, the cellular uptake of nano photosensitizers was studied. For this purpose, the targeting molecules, folic acid (FA), were covalently linked to UCNPs-ZnPc nanophotosensitizers. Fig. 8a shows the target staining of the UCNPs-ZnPc/FA nanophotosensitizers. The blue (in the web version) emission came from DAPI which stained cell nucleus in HeLa cells and human alveolar adenocarcinoma (A549) cells. The green (in the web version) and red (in the web

Fig. 9. Viability of Hela cells treated with different power density of 980 nm irradiation. Standard deviations are shown (n ¼ 4).

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Fig. 10. Viability of Hela cells treated with UCNPs-ZnPc/FA at different concentrations with (0.39 W/cm2) (red) or without (blue) 980 nm irradiation. Standard deviations are shown (n ¼ 4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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autofluorescence confirms that the UCNPs-ZnPc/FA nanophotosensitizers can be used for high-contrast luminescence imaging of cells in vitro. When the FA receptors on the cancer cell membrane were saturated by free FA before incubating with the nanophotosensitizers, few UCNPs-ZnPc/FA nanoconjugates were stained in the cancer cells (Fig. 8a, middle), which might be due to the nonspecific adsorption of UCNPs-ZnPc/FA. Furthermore, there was no significant morphology change of the cancer cells, suggesting a good biocompatibility and high security of the targeted UCNPs-ZnPc/FA nanophotosensitizers for bioimaging under NIR excitation with a low power density of 0.19 W/cm2. To further verify the specificity of the UCNPs-ZnPc/FA nanophotosensitizers, A549 cells, which are poor in expressing the folate receptor, were used for negative control (Fig. 8a, bottom). In this case only a little UCNPs were observed in cells, revealing the high bio-specificity of the constructed UCNPs-ZnPc/FA nanophotosensitizers. Moreover, to demonstrate the image-guided PDT, the morphological changes of Hela cells are analyzed as shown in Fig. 8b. It can be observed that Hela cells are spindle in morphology and clear in contour of nucleus, indicating that the cells are viable (Fig. 8b(II)). However, after irradiation of 0.39 W/cm2 of 980 nm laser for 15 min and postincubation for 24 h, the cell pseudopod disappears and the cell morphology is not clear, indicating the viability of Hela cells decreased (Fig. 8b(IV)). The result suggests that the UCNPs-ZnPc/FA

Fig. 11. Representative photos of mice and liver tumor before and after various treatments, and photos of tumor tissue were obtained after 14 days.

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Fig. 12. The growth of Hepa1-6 tumor (a) and body weight (b) in different groups after treatment.

nanophotosensitizers have the potential in applications of imageguided PDT. The overheating effect induced by 980 nm laser on cell viability was checked before PDT treatment. Hela cells were exposed to irradiation of NIR laser of 980 nm for 10 min with different powder density. The cell viability was determined from the 3-(4,5dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, as shown in Fig. 9. Within the error allowed, the power density, which was less than 0.39 W/cm2, had a negligible effect on the cell viability. When HeLa cells were exposed to NIR laser at relatively high power density of more than 1.23 W/cm2, the overheating effect became obvious, leading to rapid declined of the cells. Under this guidance, the photodynamic capabilities of the covalently assembled UCNPs-ZnPc/FA nanophotosensitizer were studied by incubating Hela cells with UCNPs-ZnPc/FA at different concentrations. The relationship between concentration and toxicity is shown in Fig. 10 without and with irradiation of 980 nm under 0.39 W/cm2. No significant decrease in cell viability was observed in the control test when the concentration of UCNPs-

ZnPc/FA is lower than 200 mg/mL. The toxicity, however, becomes only non-negligible when the concentration is higher than 200 mg/ mL. When HeLa cells were exposed to NIR laser at a relatively low intensity of 0.39 W/cm2, the cells viability declined rapidly with an increase in the concentration of UCNPs-ZnPc/FA. After confirming the image-guided PDT in vitro, we further investigated the NIR light induced PDT effects in vivo. As a proof of principle experiments, the Hepa1-6 tumor-bearing C57/6J mice were intratumoral injection of UCNPs-ZnPc/FA (at its optimal loading proportion). The Hepa1-6 tumor model was established by subcutaneously inoculating Hepa1-6 cells (3  106) into the upper axillary fossa in the mice (n ¼ 6). The mice were investigated when the tumor grew to a diameter of 4e6 mm. 100 ml of saline or UCNPsZnPc/FA (10 mg/ml) was injected into each Hepa1-6 tumor-bearing mouse in intratumoral direct injection for PDT. The mice were randomly assigned into four groups treated with different injections as follows: (1) group 1 received only intratumoral direct injection of the saline (the control group, n ¼ 6); (2) group 2 received intratumoral direct injection of the saline with NIR laser

Fig. 13. H&E stained images of (a) tumor, (b) heart, liver, spleen, lung, kidney collected from different groups.

L. Xia et al. / Biomaterials 35 (2014) 4146e4156

irradiation (n ¼ 6); (3) group 3 received intratumoral direct injection of the UCNPs-ZnPc/FA (n ¼ 6); (4) group 4 received intratumoral direct injection of the UCNPs-ZnPc/FA with NIR laser irradiation (n ¼ 6). The tumors were irradiated with 980 nm laser (0.39 W/cm2) for 15 min. To avoid any tissue damage by heating, the laser treatment was done with exposure of 980 nm laser light for 3 min per interval for 3 min. After treatment, relative tumor volume and body weight of mice were monitored every day over two weeks. The tumor volume of the group 4 mice increased much slower than other groups. As shown in Fig. 11, the subcutaneously injected Hepa1-6 cells in groups 1, 2 and 3 grew into a diameter of 10e15 mm solid tumors in two weeks after injection, while the growth of the tumor cells in group 4 was significantly inhibited. The group 4 presented significantly reduced tumor volume within two weeks and the tumor inhibitory ratio was calculated to be approximately 80.1% in comparison with group 1. The mice treated with saline and UCNPs-ZnPc/FA without light irradiation did not show any therapeutic effect, as expected. The tumor volume of the mice treated with 980 nm laser irradiation alone did not shown significant difference from that of the untreated group 1. The results coincide with the apoptosis of Hela cell treated with 980 nm irradiation alone. Body weight and survive rate usually reflect the health condition of the treated mice. As shown in Fig. 12, for the control group, relatively slow growth of their body weight which indicates the living quality of the mice is compromised by the tumor burden. For the PDT treated group, their body weight gradually increases during 14 days, implying that systemic toxicity is minimal in these mice. All these demonstrate that UCNPs-ZnPc/FA with 980 nm irradiation has an obvious inhibitory effect on tumor growth. The histological analysis on tumor, heart, liver, spleen, lung, and kidney was carried out in different treatment groups after 14 days of post-treatment. It can be clearly observed in Fig. 13a that the morphology, size and staining of the tumor cells in saline group are at variance, and mitotic figures are seen in most nuclei. It is similar to 980 nm laser group and UCNPs-ZnPc/FA group. However, markedly increased apoptotic and necrotic tumor cells were observed in all PDT treatment groups. Histological analysis shown in Fig. 13b reveals no pathological changes in the heart, lung, kidney, liver or spleen. Hepatocytes in the liver samples were found normal. No pulmonary fibrosis was detected in the lung samples. The glomerulus structure in the kidney section was observed clearly. Necrosis was not found in any of the histological samples analyzed. These results clearly demonstrate the potential clinical applicability of UCNPs-ZnPc/FA as PDT agents. 4. Conclusions We have constructed UCNPs-ZnPc/FA nanophotosensitizers, which has highly efficient single oxygen generation and safety. To improve the PDT efficacy, upconversion emission intensity of 660 nm was modulated by doping with Yb3þ ions with 25% of concentration of, and ZnPc was covalently bonded to UCNPs. Due to the high 1O2 generation, overheating effect was avoided since the laser radiation dose was able to lower to 351 J/cm2 with power density of 0.39 W/cm2, and the tumor inhibitory rate of 80.1% was reached following image-guided PDT of liver tumor. Neither pathological changes, nor inflammation was observed in various organs of tumor-bearing mice after in vivo PDT treatment following intratumoral direct injection. Acknowledgments This work was financially supported by NSF of China (11174277, 11374297, 61275202, 21304084 and 51372096), Joint research program between CAS of China and KNAW of the Netherlands, the IOP

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program of the Netherlands, and John van Geuns foundation. Prof. X.G. Kong thanks Prof. Chen L. and Prof. Li J. (Basic Medical Sciences, Jilin University.) for discussion and analysis of histological analysis.

Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2014.01.068.

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An upconversion nanoparticle--Zinc phthalocyanine based nanophotosensitizer for photodynamic therapy.

Recent advances in NIR triggering upconversion-based photodynamic therapy have led to substantial improvements in upconversion-based nanophotosensitiz...
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