Acta Biomaterialia xxx (2015) xxx–xxx

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b-Ga2O3:Cr3+ nanoparticle: A new platform with near infrared photoluminescence for drug targeting delivery and bio-imaging simultaneously Xin-Shi Wang a, Jun-Qing Situ a, Xiao-Ying Ying a, Hui Chen a, Hua-fei Pan c, Yi Jin b,⇑, Yong-Zhong Du a,⇑ a

College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, PR China National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Traditional Chinese Medicine, 56 Yangming Road, 330006, PR China c Intensive Care Unit, The First Affiliated Hospital, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, PR China b

a r t i c l e

i n f o

Article history: Received 29 October 2014 Received in revised form 2 April 2015 Accepted 8 April 2015 Available online xxxx Keywords: b-Ga2O3:Cr3+ Nanoparticles Bio-imaging Drug carrier Hyaluronic acid

a b s t r a c t Multifunctional nanoparticles which integrate the therapeutic agents and bio-imaging agents into one carrier are emerging as a promising therapeutic platform. Herein, GaOOH:Cr3+ was firstly synthesized using improved hydrothermal method (atmospheric pressure, 95 °C), and by manipulating the pH of the reaction medium, GaOOH:Cr3+ with different sizes (125.70 nm, 200.60 nm and 313.90 nm) were synthesized. Then b-Ga2O3:Cr3+ nanoparticles with porous structures were developed as a result of the calcination of GaOOH:Cr3+. The fabricated, porous b-Ga2O3:Cr3+ nanoparticles could effectively absorb doxorubicin hydrochloride (DOX) (loading rate: 8% approximately) and had near infrared photoluminescence with a 695 nm emission. Furthermore, b-Ga2O3:Cr3+ nanoparticles were coated with L-Cys modified hyaluronic acid (HA-Cys) by exploiting the electrostatic interaction and the cross-link effect of disulfide bond to improve the stability. The DOX loaded HA-Cys coated b-Ga2O3:Cr3+ nanoparticles (HA/bGa2O3:Cr3+/DOX) showed an oxidation–reduction sensitive drug release behavior. The HA-Cys coated b-Ga2O3:Cr3+ nanoparticles showed a low cytotoxicity on MCF-7 and Hela cell lines. The cellular uptake of HA/b-Ga2O3:Cr3+/DOX using the near infrared photoluminescence of b-Ga2O3:Cr3+ nanoparticles and the fluorescence of DOX demonstrated the HA/b-Ga2O3:Cr3+/DOX could internalize into tumor cells quickly, which was affected by the size and shape of b-Ga2O3:Cr3+nanoparticles. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction With the development of nanotechnology and biotechnology, multifunctional nanoparticles which can not only yield a targeting and long circulating drug/gene delivery, but also be exploited as bio-imaging agents are designed as a new area of cancer theranostic. Nanodrug delivery system such as liposomes, protein/virusbased nanoparticles, dendrimers, micelles, nanogold, mesoporous silica and carbon nanotuble modified with targeting groups (ligands or antibodies) can change the bio-distribution of drug and achieve tumor targeting therapy [1–4]. These nanodrug delivery systems have been investigated for several decades as a promising platform for personalized diagnose and treatment, but few clinical applications have been found due to their ambiguous metabolism and bio-distribution. Therefore, it is necessary to ⇑ Corresponding authors. Tel./fax: +86 57188208439. E-mail addresses: [email protected] (Y. Jin), [email protected] (Y.-Z. Du).

monitor the fate of the drug delivery system in vivo. Combining the delivery system with fluorescence probes is an advisable strategy. Fluorescent markers include organic and inorganic probes. Organic fluorescent probes, such as fluorescein isothiocyanate (FITC), Cy5.5 have poor chemical stability and are easily photobleached. Inorganic nanoprobes, such as nanogold, quantum dots (QDs) and superparamagnetic iron oxide nanoparticle have attracted great interest in recent years due to their unique characteristics. Nanogold, due to its biocompatibility, high X-ray absorption and photo-thermal effect, has attracted much attention in cancer diagnoses and therapy. But as a CT contrast agent, it may lead to the harm caused by X-rays [5]. QDs are an inorganic semiconductor with a strong fluorescence emission, which are extensively applied in molecular probe, cell and small animal imaging research, but the cytotoxicity is noticeable [6,7]. Superparamagnetic iron oxide nanoparticles, used as contrast probes for magnetic resonance imaging (MRI) are insensitive

http://dx.doi.org/10.1016/j.actbio.2015.04.010 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Wang X-S et al. b-Ga2O3:Cr3+ nanoparticle: A new platform with near infrared photoluminescence for drug targeting delivery and bio-imaging simultaneously. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.010

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[8,9]. b-Ga2O3 is an important semiconductor, which has been widely applied in optoelectronic devices because of the excellent optical performance. Rare earth ion-doped b-Ga2O3 has attracted extensive attention because of its photoluminescence (PL) properties. It can radiate various photoluminescence spectra from visible to near-infrared light when doped with different foreign impurities [10–12]. However, Cr3+ doped b-Ga2O3 used not only as a fluorescent probe for bio-imaging but also a drug carrier has not been reported. To achieve an active tumor targeting, surface modification of the carrier is necessary. The normal strategy is to conjugate tumor targeting groups such as folic acid (FA), transferrin (Tf), galactose (Gal) and epidermal growth factor (EGF) onto the surface of drug carriers. Hyaluronic acid (HA) is a negatively charged, linear polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine [13]. Because of its biodegradability, biocompatibility and non-immunogenicity, HA has been extensively investigated for biomedical applications such as gene/drug carrier [14,15], tissue engineering [16,17] and regenerative medicine [18]. Particularly, HA can bind to the CD44 receptor, which is overexpressed in various cancer cells, such as HBL-100, HCT-116, HT-29 and Hela cells [19,20]. But HA can be easily degraded by hyaluronidases-2 in the surface of cell and hyaluronidases-1 in the lysosome [21]. Hence, chemical modification is necessary to prolong the life time of HA and improve its stability in vivo. Disulfide-cross-linking is one of the suitable strategies [22–24]. In this study, homogeneous, porous b-Ga2O3:Cr3+ nanoparticle with different shape and size was synthesized to load antitumor drug DOX. To improve the stability and targeting ability of nanoparticles, b-Ga2O3:Cr3+ nanoparticles were then coated with L-Cys

modified HA through electrostatic interaction. After the investigations such as photoluminescence spectra, drug loading and in vitro drug release, the cytotoxicity and cellular uptake were further conducted using MCF-7 and Hela cell lines. 2. Materials and methods 2.1. Materials Gallium oxide (Ga2O3, 99.99%), sodium hyaluronate (HA, 95%), Sodium acetate trihydrate (AR), glacial acetic acid (AR),

L-Cysteine

(L-Cys, 98%), 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride (EDAC, 98.5%), N-hydroxysuccinimide (NHS, 98%), hydrogen peroxide (H2O2, 30%), chromic nitrate (Cr(NO3)39H2O, 99.95%), glutathione (GSH, 98%) were purchased from Aladdin (Shanghai, China). Doxorubicin hydrochloride (DOX) was purchased from Dalian Meilun Biology Technology Co. Ltd (Dalian, China). PE Mouse Anti-Human CD44 kit was purchased from BD Biosciences (America). All the materials were used without purification. Hela, MCF-7 cells were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). RPMI 1640 culture medium with penicillin (100 unit/ml) and streptomycin (100 unit/ml) were purchased from Jinuo Biomedical Technology Co. Ltd (Hangzhou, China). Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biological Technology Co. Ltd (Huzhou, China). Cells were cultivated in RPMI 1640 with 10% FBS, at 37 °C with 5% CO2. 2.2. Preparation and characterizations of GaOOH:Cr3+and bGa2O3:Cr3+nanoparticles The GaOOH:Cr3+ nanoparticles were synthesized by heating and stirring in water bath. Firstly, Ga2O3 (1.5 g) was dissolved in 50 ml

HCl (2 M) through stirring and heating. 40 g Sodium acetate trihydrate was dissolved in 800 ml H2O, and then 0.8 ml Cr(NO3)39H2O (0.1 M) was added with stirring. The above two solutions were homogeneously mixed under magnetic stirring at room temperature for 15 min, and then the pH was adjusted to 4.5, 5.5 or 7.0 using glacial acetic acid or NaOH (2 M), finally the reaction system was added up to 1000 ml with water. The reaction solution was heated and stirred at 70 °C for 1 h and then at 95 °C for another 4 h. The obtained white precipitate was separated by centrifugation and collected after being washed with deionized water for 3 times. At last, the GaOOH:Cr3+ was precalcined at 600 °C for 3 h and then calcined at 950 °C for another 3 h to prepare b-Ga2O3:Cr3+ . The shape and crystal structure of GaOOH:Cr3+ and b-Ga2O3:Cr3+ were studied by transmission electron microscopy (TEM; JEM-1200EX, Japan) and X-ray diffraction (X’Pert PRO, Panalytical), respectively. Average particle size of the GaOOH:Cr3+ was determined by Zetasizer Nano (S90 Malvern). Thermogravimetric analysis (TGA) of GaOOH:Cr3+ was performed using Thermal Analysis (DSCQ1000, AT America) with a heating rate of 10 °C/min from room temperature to 800 °C in air. Zeta potential of b-Ga2O3:Cr3+ dispersed in Milli-Q water was determined by Zetasizer Nano ZS (NANO-ZS90, Malvern). The excitation and emission spectra of nanoparticles were acquired by fluorescence spectrophotometer (FLS920, Edinburgh Instruments, England). 2.3. Synthesis and characterization of HA-Cys L-Cysteine modified HA (HA-Cys) was synthesized through the linkage of amide bond. Briefly, HA (0.20 g) was added to 50 ml H2O and stirred for 1 h until sufficient dissolution. EDAC (0.48 g) and NHS (0.29 g) were added to the solution (pH was adjusted to 5.5) and stirred for 20 min. Then, L-Cysteine (0.20 g) was added (pH was adjusted to 6.0). After stirring at room temperature for 4 h, the mixture was dialyzed in 0.9% NaCl solution for 4 times followed by distilled water. Finally, the product was lyophilized and stored at 4 °C. The structure of HA-Cys was confirmed by 1H NMR spectra measured (HA or HA-Cys was dissolved in D2O) by nuclear magnetic resonance (Bruker AVIII 500 Mspectrometer, Fällan-den, Switzerland).

2.4. Synthesis and characterization of HA/b-Ga2O3:Cr3+ For the synthesis of HA/b-Ga2O3:Cr3+, b-Ga2O3:Cr3+ (100 mg) was dispersed in 3 ml purified water and then HA-Cys (30 mg) was added. After stirring for 2 h, 100 ll H2O2 was added into the solution and continuously stirred for another 2 h. The product was collected by centrifuging (12,000 rpm, 20 min) followed by washing and then lyophilized. Zeta potential of HA/b-Ga2O3:Cr3+dispersed in Milli-Q water was measured by Zetasizer Nano ZS (NANO-ZS90, Malvern). The mass of HA-Cys coated on the surface of b-Ga2O3:Cr3+ nanoparticles was measured by TGA with heating rate of 10 °C/min from room temperature to 800 °C in air. Before TGA analysis, b-Ga2O3:Cr3+ nanoparticles and HA/b-Ga2O3:Cr3+ were maintained at 60 °C for 20 h to remove moisture. 2.5. Synthesis and characterization of HA/b-Ga2O3:Cr3+/DOX For the synthesis of b-Ga2O3:Cr3+/DOX, b-Ga2O3:Cr3+ nanoparticles were mixed with DOX solution (PBS, pH 7.4, 0.01 M) followed by ultrasonication for 0.5 h. After stirring for 12 h with light protection, the solution was centrifuged and the sediment was washed with H2O for 3 times followed by lyophilization. The supernatant was collected to determine drug loading rate. The absorption

Please cite this article in press as: Wang X-S et al. b-Ga2O3:Cr3+ nanoparticle: A new platform with near infrared photoluminescence for drug targeting delivery and bio-imaging simultaneously. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.010

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spectroscopy at 480 nm of the supernatant was determined by UV–VIS. The drug-loading rate was calculated by the following formula (Eq. (1)):

Loading rate ¼ ðm1  m2 Þ=ðm1  m2 þ m3 Þ  100%

ð1Þ

m1 is the amount of total DOX, m2 is the amount of unloaded DOX in the supernatant, m3 is the amount of b-Ga2O3:Cr3+ nanoparticles. For the synthesis of HA/b-Ga2O3:Cr3+/DOX, b-Ga2O3:Cr3+/DOX was dispersed in HA-Cys solution (10 mg/ml) (b-Ga2O3:Cr3+/DOX to HA: w/w = 4:1). After stirring in open air for 4 h, the HA/bGa2O3:Cr3+/DOX nanoparticles were collected by centrifugation and washed with water. The supernatant was collected to measure leaked drug by using UV–VIS absorption spectroscopy at 480 nm. Drug loading rate of HA/b-Ga2O3:Cr3+/DOX was calculated by the following formula (Eq. (2)):

Loading rate ¼ ðm4  m5 Þ=m6  100%

ð2Þ

m4 is the amount of DOX in b-Ga2O3:Cr3+/DOX, m5 is the amount of leaked DOX from b-Ga2O3:Cr3+/DOX, m6 is the total mass of HA/bGa2O3:Cr3+/DOX after lyophilization. 2.6. Cytotoxicity assay of HA/b-Ga2O3:Cr3+and HA/b-Ga2O3:Cr3+/DOX The cytotoxicity assay was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) reduction assays. Hela and MCF-7 cell were seeded in 96-well plates and incubated for 24 h, respectively. The cells were exposed to a series of concentrations of free DOX, HA/b-Ga2O3:Cr3+, HA/b-Ga2O3:Cr3+/ DOX for an additional 24 h. Blank wells were also added to corresponding concentrations of nanoparticles. At the end of the incubation time, 32 ll of MTT (5 mg/ml) was added to each well. After an additional 4 h incubation, the mixture was replaced with 200 ll DMSO and shaken for 15 min. The solution was transferred to a new 96-well plate to measure the optical density (OD) at 570 nm by microplate reader (Multiskan MK3, Thermo Scientific, USA). The cell viability was calculated by the following formula (Eq. (3)):

Cell viability ¼ ðOD1  ODblank Þ=ðODcontrol  ODblank Þ  100%

ð3Þ

OD1 : OD of free DOX; HA=b  Ga2 O3 : Cr3þ or HA=b  Ga2 O3 : Cr3þ =DOX: 2.7. In vitro DOX release For the in vitro drug release tests, 5 mg of HA/b-Ga2O3:Cr3+/DOX nanoparticles was dispersed in 3 ml release media (PBS of pH 5.5 + 0 mM GSH, pH 5.5 + 10 mM GSH or pH 7.4 + 10 lM GSH) at 37 °C with 100 rpm shaking. At predetermined time intervals, absorbance of centrifugate (8000 rpm, 5 min) was measured at 480 nm. The sediment was dispersed in another 3 ml release media for further release tests. All drug release tests were performed three times. 2.8. Cellular uptake For the cell active selective imaging, MCF-7 and Hela cells were seeded in 6-well at a density of 2  104 cells per chamber and allowed to attach overnight. The cells were then treated with HA/b-Ga2O3:Cr3+/DOX (at a final nanoparticle concentration of 150 lg/mL) for different time periods. The nuclei were labeled with hochest33342 and fixed with 4% paraformaldehyde. After washing the cells three times with PBS, the cells were observed using confocal laser scanning microscopy (LSCM, LSM-510, Zeiss, Germany). The wavelength of exciting light of hochest33342 and b-Ga2O3:Cr3+ was 405 nm and 635 nm, respectively.

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To investigate the effect of HA and CD44 receptor interaction to the cellular uptake of nanoparticles, Hela cells were incubated with free HA (4 mg/ml) for 1 h before adding HA/b-Ga2O3:Cr3+/DOX. After incubating for designated intervals, Hela cells were collected and analyzed by LSCM and FCM. For the analysis of cellular uptake of different sizes of HA/bGa2O3:Cr3+/DOX, after being exposed to HA/b-Ga2O3:Cr3+/DOX (the final DOX net weight of the nanoparticle was 2 lg/ml perwell) for 4 h, the cells were washed with PBS and digested with trypsin enzyme, then collected and fixed with 4% paraformaldehyde. The obtained cells were analyzed by Flow Cytometer (FCM, FC500MCL, BECKMAN COULTER, AMERICA). And Hela cells were cultivated in 6 well plates for LSCM. The wavelength of exciting light of DOX was 488 nm. 2.9. Statistical analysis All the data represent mean values ± SD (n = 3). Statistical analysis was performed using Student’s t test and statistical significance was assigned at p < 0.05 (95% confidence level). 3. Results and discussion 3.1. Synthesis and characterization of GaOOH:Cr3+and b-Ga2O3:Cr3+ Hydrothermal method is a common synthetic method for GaOOH, however, due to the need of the special reaction vessel (reaction kettle), and the violent reaction condition (>200 °C) [25,26], the application of this method is restricted to small scale. In this study, GaOOH:Cr3+ was synthesized using improved hydrothermal method with moderate reaction condition (atmospheric pressure, 95 °C) and larger reaction capacity. Firstly, GaOOH:Cr3+ with different shape and size were obtained by manipulating the pH of the reaction medium. As shown in Fig. 1, the spindle-like GaOOH:Cr3+ with a particle size of 125.70 ± 35.70 nm (G10 ), the GaOOH:Cr3+ nanorod with a particle size of 200.60 ± 51.68 nm (G20 ) and GaOOH:Cr3+ nanorod with a particle size of 313.90 ± 116.20 nm (G30 ) were synthesized under pH 4.5, pH 5.5 and pH 7.0, respectively. GaOOH is an important precursor for the preparation of b-Ga2O3 and has a decisive effect on the shape and size of the final b-Ga2O3. Then the final product b-Ga2O3:Cr3+ with different shape and size was obtained by the calcination of GaOOH:Cr3+ having variational shape and size at 950 °C for 3 h. The high crystallinity of GaOOH:Cr3+ and b-Ga2O3:Cr3+ was reflected by the sharp and narrow peaks in XRD as shown in Fig. 2A. All the reflections were indexed to GaOOH (JCPDS: 060180) and b-Ga2O3 (JCPDS: 41-1103), respectively. Notably, the minor doped Cr3+ showed no influence on the crystal structure of b-Ga2O3. The weight loss of the conversion from GaOOH:Cr3+ to b-Ga2O3:Cr3+ was measured at 13.95% (Fig. 2B), which was similar with previous report, and it was higher than theoretical value (8.76%) [27,28]. This may be attributed to the water on the surface of nanoparticle and in the crystal lattice, and hydroxyl groups which were bonded on the surface [29]. b-Ga2O3:Cr3+ with the same morphology as GaOOH:Cr3+ and porous structure were observed from Fig. 2C. The excitation and emission spectra of b-Ga2O3:Cr3+ nanoparticles with different size and shape are displayed in Fig. 2D. The broad emission band (690–850 nm) was attributed to 4T2 ? 4A2 transition of Cr3+, which indicated that Cr3+ was doped into the b-Ga2O3 crystal lattice host [12]. The emission spectra were in the near infrared window (650–900 nm), and the biological tissues have low absorption in this wavelength [30]. The photoluminescence intensity (excitation wavelength: 635 nm) of b-Ga2O3:Cr3+varied with the

Please cite this article in press as: Wang X-S et al. b-Ga2O3:Cr3+ nanoparticle: A new platform with near infrared photoluminescence for drug targeting delivery and bio-imaging simultaneously. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.010

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Fig. 1. TEM photographs and size distribution of GaOOH:Cr3+prepared by different pH (G10 : pH = 4.5, G20 : pH = 5.5 and G30 : pH = 7.0).

Fig. 2. (A) XRD pattern of GaOOH:Cr3+ and b-Ga2O3:Cr3+; (B) TGA of GaOOH:Cr3+; (C) TEM images of b-Ga2O3:Cr3+acquired by calcination of GaOOH:Cr3+ (G1, G2, G3); (D) PL characteristic of b-Ga2O3:Cr3+ (G1, G2, G3). The left is the excitation spectrum (emission wavelength: 720 nm), and the right is the emission spectrum (excitation wavelength: 635 nm).

shape and size. This may be explained by the fact that the amount of Cr3+ doped into the b-Ga2O3 varied with the pH of the reaction medium and more sites of Ga3+ were occupied by Cr3+, the stronger of emission intensity was in a relatively low concentration range [31]. To verify this, b-Ga2O3 with different contents of doped Cr3+ were synthesized, and it was found that the fluorescence intensity

was enhanced with the increase of Cr3+ (Figs. S1 and S2). Besides, near infrared persistent luminescence, which has been reported to be applied for bio-imaging [32,33], was also found in the bGa2O3:Cr3+ nanoparticles. After being activated by UV for 10 min, the luminescence could maintain more than 24 h (Fig. S3), which was longer than that in the previous report [34]. However, the

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persistent luminescence signal lasted no more than 0.5 h in a nude mouse (intravenous injection, 1 mg b-Ga2O3:Cr3+), which was too short to be used for the monitor of the fate of the b-Ga2O3:Cr3+ in vivo. Therefore, the fluorescence signal in near infrared window of the b-Ga2O3:Cr3+ instead of the persistent luminescence was collected for bio-imaging in the following experiments. 3.2. Preparation and characterization of HA/b-Ga2O3:Cr3+ To achieve the purpose of selectively targeting cancer cells in which CD44 receptor is over expressed, b-Ga2O3:Cr3+ nanoparticles coated with HA-Cys were prepared (HA/b-Ga2O3:Cr3+). Firstly, HA modified with L-Cysteine was synthesized through reaction between carboxylic acid groups in HA and amine groups in L-Cysteine 1

(Fig. 3A). The structure of HA-Cys was confirmed by H NMR (Fig. 3B and C). Characteristic peak of methylene at 2.93 ppm was found in the 1H NMR spectrum of HA-Cys while there was no chemical shift to signify methylene of HA. These results demonstrated the successful linkage between HA and L-Cysteine

[35]. As shown in Fig. 4A, the average zeta potential of three b-Ga2O3:Cr3+nanoparticles without HA-Cys dispersed in Milli-Q water was 27.80 ± 1.25 mV (G1), 24.53 ± 1.50 mV (G2) and 31.47 ± 0.51 mV (G3), respectively. However, the zeta potential of b-Ga2O3:Cr3+ shifted greatly, after being coated with HA-Cys. The positive potential of the b-Ga2O3:Cr3+may be a result of the corresponding unsaturated Ga3+ ions in the b-Ga2O3:Cr3+ nanoparticles due to the oxygen vacancies [36,37], while the shift of the potential of b-Ga2O3:Cr3+ after being coated by HA-Cys may be a result of the absorption of HA-Cys on the surface of b-Ga2O3:Cr3+, since HA is a negatively charged polysaccharide. Furthermore, Fig. 4B shows all the three uncoated b-Ga2O3:Cr3+ aggregated and precipitated within 1 h in PBS (pH 7.4). The HA/b-Ga2O3:Cr3+ with largest size (H/G3) was precipitated after 10 h because of the gravity while other two kinds of HA/b-Ga2O3:Cr3+ with smaller size (H/G1 and

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H/G2) were still well dispersed in pH 7.4 PBS. The dramatically, improved stability of b-Ga2O3:Cr3+ coated by HA-Cys may be due to the coat of HA-Cys which prevented the nanoparticle from aggregating. The amount of HA-Cys coated on the surface of b-Ga2O3:Cr3+ nanoparticle was determined by TGA. As shown in Fig. 4C, the weight loss of H/G1 was not significant until the temperature was higher than 200 °C and when the temperature continued to increase (>400 °C), the weight of HA/b-Ga2O3:Cr3+ was constant. Similar weight loss curves of H/G2 and H/G3 were observed while the weight of the G3 kept the same level when the temperature shifted from room temperature to 800 °C. These results can be explained by the fact that b-Ga2O3:Cr3+ nanoparticles would not lose weight in the TGA theoretically, since it had been calcined for 3 h in the preparation process, while HA-Cys on the surface of HA/b-Ga2O3:Cr3+ was carbohydrate and began to decompose at 200 °C (Fig. S4), which was consistent with the previous reports [38]. 3.3. Preparation and characterization of HA/b-Ga2O3:Cr3+/DOX Firstly, b-Ga2O3:Cr3+/DOX was prepared by the absorption between DOX and b-Ga2O3:Cr3+. As shown in Fig. 5A, the DOX loading rate increased with the increase of DOX content, while the encapsulation efficiency decreased with the rise of the DOX content. In consideration of the high encapsulation efficiency and appropriate loading rate, the weight ratio of b-Ga2O3:Cr3+: DOX was determined at 20:1. Then b-Ga2O3:Cr3+/DOX were coated with HA-Cys, while the loading rate slightly declined during the process of coating (Fig. 5B), which may be due to the leakage of DOX from b-Ga2O3:Cr3+/DOX and package of HA-Cys. As shown in Fig. 5C, without GSH, or in 10.0 lM of GSH, the drug release was slow and only about 30% of DOX was released in 10 h. In the presence of 10.0 mM GSH, the release of DOX was significantly accelerated, and approximately 60% of the loaded

Fig. 3. (A) The synthesis scheme of HA-Cys. The 1H NMR spectra of HA (B) and HA-Cys (C) dissolved in D2O.

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Fig. 4. (A) Zeta potential of b-Ga2O3:Cr3+ (G1, G2, G3) and HA/b-Ga2O3:Cr3+ (H/G1, H/G2, H/G3) nanoparticles, the results are expressed as mean ± SD (n = 3); (B) images of three HA/b-Ga2O3:Cr3+nanoparticles after placed for 1 h and 10 h; (C) TGA of b-Ga2O3:Cr3+ and HA/ b-Ga2O3:Cr3+nanoparticles.

Fig. 5. (A) Loading rate and encapsulation efficiency of the three b-Ga2O3:Cr3+(G1, G2, G3) nanoparticles; (B) loading rates of b-Ga2O3:Cr3+/DOX (G/D) and HA/b-Ga2O3:Cr3+/ DOX (H/G/D)nanoparticles (b-Ga2O3:Cr3+:DOX = 20:1/w:w), ⁄meant significant difference (p < 0.05); (C) release profile of DOX from three HA/b-Ga2O3:Cr3+/DOX nanoparticles (H/G1/D, H/G2/D, H/G3/D) under different release medium. The results are expressed as mean ± SD (n = 3).

DOX was released in 10 h (H/G3/D, this GSH-sensitive drug release behavior was also observed from H/G1/D and H/G2/D). This GSHsensitive drug release behavior may result from the facts that the coated HA-Cys on the surface of b-Ga2O3:Cr3+/DOX was crosslinked by the disulfide bond as a result of open air stirring [39]. Disulfide bonds are prone to be rapidly cleaved through thioldisulfide exchange reaction [40] especially where there is GSH. GSH is found both in plasma and intracellular, but the concentration in the plasma is usually 100–1000 times less than that in intracellular, and GSH is mainly found in the cytosol where the concentration reaches 1–11 mM [41] which meant the fabricated HA/b-Ga2O3:Cr3+/DOX could selectively release the encapsulated DOX intracellular. 3.4. Cytotoxicities of HA/b-Ga2O3:Cr3+ and HA/b-Ga2O3:Cr3+/DOX To study the biocompatibility of HA/b-Ga2O3:Cr3+, the cytotoxicity of HA/b-Ga2O3:Cr3+ against MCF-7 and Hela was conducted. As shown in Fig. 6A and B, the cell viabilities of both MCF-7 and

Hela were all above 80% even when the concentration of HA/b-Ga2O3:Cr3+ was up to 1000 lg/ml. While after being loaded with DOX, the cytotoxicity of HA/b-Ga2O3:Cr3+shifted greatly (Fig. 6C and D), which may be due to the DOX released from the carriers. 3.5. Selectively accumulate in CD44-positive tumor cells To investigate the cellular uptake of b-Ga2O3:Cr3+/DOX and HA/b-Ga2O3:Cr3+/DOX nanoparticles, Hela and MCF-7 cell lines were selected as model cells [42,43]. As shown in Fig. 7, the fluorescence intensity of HA/b-Ga2O3:Cr3+/DOX treated cells (MCF-7 and Hela) was significantly stronger than that of b-Ga2O3:Cr3+/ DOX treated groups, which may be due to the targeting ability of HA to CD44. Also, in the HA/b-Ga2O3:Cr3+/DOX treated groups, the fluorescence intensity in Hela cells was slightly stronger than that in MCF-7 cells which may be due to the slightly higher level of CD44 expressed by Hela than that of MCF-7 cells (Fig. S5). Furthermore, to confirm the targeting ability of HA, a HA blocking

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Fig. 6. Cell viabilities of MCF-7 (A) and Hela (B) cell lines after incubated with a series of concentration of three HA/b-Ga2O3:Cr3+ (H/G1, H/G2, H/G3) nanoparticles. Abscissa is the concentration of b-Ga2O3:Cr3+net weight. Cell viabilities of MCF-7 (C) and Hela (D) cells after being incubated with a series of concentrations of HA/b-Ga2O3:Cr3+/DOX (H/ G1/D, H/G2/D, H/G3/D). Abscissa is the concentration of DOX net weight. The results are expressed as mean ± SD (n = 3).

Fig. 7. LSCM images of Hela cells after being incubated with b-Ga2O3:Cr3+/DOX (G1/D) or HA/b-Ga2O3:Cr3+/DOX(H/G1/D) nanoparticles for 1, 4, 8 h. Cell nucleus was stained with hochest33342 (blue) and the red fluorescence was from b-Ga2O3:Cr3+.

experiment was conducted. As shown in Fig. 8A, after being pretreated with free HA, the fluorescence intensity was significantly weakened, and in contrast, the fluorescence intensity of HA untreated groups increased with the prolonged time, which was due to the saturation of CD44 receptors by HA [44,45]. These results were also quantitively confirmed by FCM (Fig. 8B). Using DOX as a fluorescence probe, a similar result was obtained (Fig. S6).

To study the effect of shape and size of HA/b-Ga2O3:Cr3+/DOX (H/G/D) on the cellular uptake, the cellular uptake of H/G1/D, H/ G2/D and H/G3/D was observed. As shown in Fig. 9A, after being incubated with HA/b-Ga2O3:Cr3+/DOX (the final DOX content: 2 lg/ml), the fluorescence intensity of H/G1/D in Hela cells was the weakest, and the H/G2/D had the highest fluorescence intensity, which was also confirmed by FCM (Fig. 9B). These results demonstrated the cellular uptake of HA/b-Ga2O3:Cr3+/DOX was

Please cite this article in press as: Wang X-S et al. b-Ga2O3:Cr3+ nanoparticle: A new platform with near infrared photoluminescence for drug targeting delivery and bio-imaging simultaneously. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.010

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X.-S. Wang et al. / Acta Biomaterialia xxx (2015) xxx–xxx

Fig. 8. (A) LSCM images of Hela cells after exposed to HA/b-Ga2O3:Cr3+/DOX (H/G1/D) nanoparticles for 1, 2, 4 h. Cell nucleus was stained with hochest33342 (blue) and the red fluorescence in cytoplasm was from b-Ga2O3:Cr3+; (B) quantitative cellular uptake analyzed by FCM. b-Ga2O3:Cr3+ was used as fluorescence marker.

Fig. 9. (A) LSCM images of Hela cells after being incubated with different sizes of HA/b-Ga2O3:Cr3+/DOX (H/G1/D, H/G2/D, H/G3/D) nanoparticles for 4 h. Cell nucleus was stained with hochest33342 (blue) and the green fluorescence in cytoplasm is from DOX loaded in complex; (B) quantitative cellular uptake analyzed by FCM. DOX loaded in nanoparticles was used as fluorescence marker.

affected by the shape and size of the particle. And the shape of HA/ b-Ga2O3:Cr3+/DOX had a larger effect on the cellular uptake than that of size (the spindle-like nanoparticle had a poor cellular uptake than that of nanorod). Additionally, when the influence of the shape of HA/b-Ga2O3:Cr3+/DOX was excluded, the smaller the particle was, the stronger the cellular uptake was.

4. Conclusions In summary, uniform, porous b-Ga2O3:Cr3+ nanoparticles with different size and shape were successfully synthesized. The porous b-Ga2O3:Cr3+ nanoparticles had near infrared photoluminescence and could effectively absorb antitumor drug DOX. b-Ga2O3:Cr3+

Please cite this article in press as: Wang X-S et al. b-Ga2O3:Cr3+ nanoparticle: A new platform with near infrared photoluminescence for drug targeting delivery and bio-imaging simultaneously. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.010

X.-S. Wang et al. / Acta Biomaterialia xxx (2015) xxx–xxx

coated with L-Cys modified HA had a better stability, GSH-sensitive drug release behavior and the ability of targeting CD44-positive tumor cells. All these results suggested that HA/b-Ga2O3:Cr3+/ DOX was a potential theranostic platform for bio-imaging and cancer targeting therapy. Acknowledgements This work was supported by the Scientific Research Fund of Ministry of Health-Medical Science Major Technology Fund Project of Zhejiang Province (No. WKJ2012-2-023) and National Natural Science Foundation of China (No. 81172999). Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Figs. 1, 2, 4, and 6–9, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2015.04.010. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.04. 010. References [1] Du YZ, Weng Q, Yuan H, Hu FQ. Synthesis and antitumor activity of stearate-gdextran micelles for intracellular doxorubicin delivery. ACS Nano 2010;4:6894–902. [2] Du YZ, Cai LL, Liu P, You J, Yuan H, Hu FQ. Tumor cells-specific targeting delivery achieved by A54 peptide functionalized polymeric micelles. Biomaterials 2012;33:8858–67. [3] Ying XY, Wang Y, Liang J, Yue JX, Xu CL, Lu L, et al. Angiopep-conjugated electro-responsive hydrogel nanoparticles:therapeutic potential for epilepsy. Angew Chem Int Ed 2014;53. http://dx.doi.org/10.1002/anie.201403846. [4] Mishra D, Hubenak JR, Mathur AB. Nanoparticle systems as tools to improve drug delivery and therapeutic efficacy. J Biomed Mater Res A 2013;101: 3646–60. [5] Khan MS, Vishakante GD, Siddaramaiah H. Gold nanoparticles: a paradigm shift in biomedical applications. Adv Colloid Interface 2013;199–200:44–58. [6] Jamieson T, Bakhshi R, Petrova D, Pocock R, Imani M, Seifalian AM. Biological applications of quantum dots. Biomaterials 2007;28:4717–32. [7] Probst CE, Zrazhevskiy P, Bagalkot V, Gao XH. Quantum dots as a platform for nanoparticle drug delivery vehicle design. Adv Drug Deliver Rev 2013;63:703–18. [8] Key J, Leary JF. Nanoparticles for multimodal in vivo imaging in nanomedicine. Int J Nanomed 2014;9:711–26. [9] Xie J, Lee S, Chen XY. Nanoparticle-based theranostic agents. Adv Drug Deliver Rev 2010;62:1064–79. [10] Liu GC, Duan XC, Li H, Liang D. Preparation and photoluminescence properties of Eu-doped Ga2O3 nanorods. Mater Chem Phys 2008;110:206–11. [11] Zhou SF, Feng GF, Wu BT, Jiang N, Xu SQ, Qiu JR. Intense infrared luminescence in transparent glass-ceramics containing b-Ga2O3:Ni2+ nanocrystals. J Phys Chem C 2007;111:7335–8. [12] Nogales E, Garcia JA, Méndez B, Piqueras J. Red luminescence of Cr in Ga2O3 nanowires. J Appl Phys 2007;101:033517. [13] Xu X, Jha AK, Harrington DA, Farach-Carson MC, Jia XQ. Hyaluronic acid-based hydrogels: from a natural polysaccharide to complex networks. Soft Matter 2012;8:3280–94. [14] Bauhuber BS, Hozsa C, Breunig M, Gopferich A. Delivery of nucleic acids via disulfide-based carrier systems. Adv Mater 2009;21:3286–306. [15] Choi KY, Yoon HY, Kim JH, Bae SM, Park RW, Kang YM, et al. Smart nanocarrier based on pegylated hyaluronic acid for cancer therapy. ACS Nano 2011;5: 8591–9. [16] Collins MN, Birkinshaw C. Hyaluronic acid based scaffolds for tissue engineering–a review. Carbohyd Polym 2013;92:1262–79. [17] Zorlutuna P, Annabi N, Camci-Unal G, Nikkhah M, Cha JM, Nichol JW, et al. Microfabricated biomaterials for engineering 3D tissues. Adv Mater 2012;24:1782–804.

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Please cite this article in press as: Wang X-S et al. b-Ga2O3:Cr3+ nanoparticle: A new platform with near infrared photoluminescence for drug targeting delivery and bio-imaging simultaneously. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.010