Chitosan-based core-shell nanomaterials for pH-triggered release of anticancer drug and near-infrared bioimaging

Accepted Manuscript Title: Chitosan-based core-shell nanomaterials for pH-triggered release of anticancer drug and near-infrared bioimaging Author: Lianjiang Tan Ran Huang Xiaoqiang Li Shuiping Liu Yu-Mei Shen Zhifeng Shao PII: DOI: Reference:

S0144-8617(16)31158-4 http://dx.doi.org/doi:10.1016/j.carbpol.2016.09.092 CARP 11619

To appear in: Received date: Revised date: Accepted date:

3-6-2016 28-9-2016 29-9-2016

Please cite this article as: Tan, Lianjiang., Huang, Ran., Li, Xiaoqiang., Liu, Shuiping., Shen, Yu-Mei., & Shao, Zhifeng., Chitosan-based core-shell nanomaterials for pHtriggered release of anticancer drug and near-infrared bioimaging.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.09.092 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Chitosan-based core-shell nanomaterials for pH-triggered release of anticancer drug and near-infrared bioimaging

Lianjiang Tana,*, Ran Huang b, Xiaoqiang Li c, Shuiping Liu c, Yu-Mei Shena,*, Zhifeng Shaod

a

Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of

Education), Shanghai Jiao Tong University, Shanghai 200240, China. b State

Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology,

Shanghai Jiao Tong University, Shanghai 200240, China. c

Key Laboratory of Eco-Textiles, Ministry of Education and College of Textile & Clothing,

Jiangnan University, Wuxi 214122, China. d

Bio-ID Center, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai

200240, China.

*Corresponding

author.

Tel:

+86-21-34204561.

E-mail:

[email protected]

and

[email protected].

HIGHLIGHT:



Modified chitosan exhibits pH-responsive structural transformation.



Chitosan-based core-shell nanospheres can release DOX triggered by lowered pH.



The NIR-fluorescent nanospheres can be monitored in real time by NIR imaging.



DOX release from nanospheres can be activated in tumor cells for cancer therapy. 1

Abstract As a naturally-abundant biopolymer, chitosan (CS) exhibit pH-sensitive structural transformation within a narrow pH range. Integrating hydrophobic groups to CS molecules gives modified CS polymers with more adjustable pH responsiveness. In this paper, near-infrared (NIR) photoluminescent Ag2S QDs capped by long-chain carboxylic acid were synthesized and then conjugated with CS via esterification reaction. The anticancer drug doxorubicin (DOX) has an affinity for the hydrophobic oleoyl groups and was entrapped by them to produce Ag2S(DOX)@CS nanospheres. A variety of experiments were performed to characterize the nanospheres. In vitro and in vivo experiments showed that the nanospheres can release DOX at lowered pH in tumor cells and have high antitumor efficacy. In addition, the strong NIR signal derived from the encapsulated Ag2S QDs makes real-time monitoring of the nanosphere distribution in a body possible. This study provides a new CS-based nanocomposite drug carrier for efficient cancer therapy.

Key words: chitosan; core-shell nanospheres; pH-triggered drug release; antitumor; NIR bioimaging

1. Introduction Cancer has been one of the leading causes of deaths across the world (Ferlay et al., 2012). Among cancer therapeutic methods, chemotherapy is the most frequently used one for its high efficiency. Nanoscale drug delivery is attracting increasing attention because of their unique properties including large surface area and high affinity for tissues (Geszke-Moritz & Moritz, 2013; Qiao et al., 2014). In order to develop efficient drug delivery systems for cancer therapy, a variety of nanomaterials have been designed as drug carriers (Hubbell & Chilkoti, 2012; Li, 2015; Linderoth, 2009; Wang et al., 2011; Yang, 2011; Zhou, Huang, Liu, Zhu & Yan, 2010), where the anticancer drugs are loaded by either physical entrapment or chemical conjugation. Nanosized drug carriers with pH-responsiveness have been studied extensively in view of the acidic 2

environment in cancer cells (Li et al., 2013; Lv et al., 2014). Fast response to pH changes plays a pivotal role in efficient drug release. On the other hand, real-time monitoring of drug delivery vehicles in the body is advantageous for understanding distribution and targeted accumulation of the drug carriers. As a cationic polysaccharide, chitosan (CS) has been widely used for a variety of biomedical applications (Flitney, Megson, Thomson, Kennovin & Butler, 1996; Jayasree, Sasidharan, Koyakutty, Nair & Menon, 2011; Tan, Wan, Li & Lu, 2012; Yang et al., 2015). The pKa of CS is 6.0-6.5 in aqueous media (Tan, Wan & Li, 2013), and the charged state and physiochemical properties of CS are significantly influenced by the ambient environmental pH (Rinaudo, 2006). CS was found to form dissociated precipitates in aqueous media at physiological pH of 7.4 due to rapid local aggregation of CS polymeric chains (Chen, Chiu, Chen, Ho & Sung, 2011). Sung’s group fabricated a comb-like associating polyelectrolyte by conjugating a hydrophobic palmitoyl group onto the free amine groups of CS (Montembault, Viton & Domard, 2005; Rinaudo, 2006). By simply adjusting the environmental pH within a narrow range and thereby balancing charge repulsion and hydrophobic interaction, the chain conformation of the associating polyelectrolyte may be controlled (Chen, Chiu, Chen, Ho & Sung, 2011). As emerging semiconductor nanocrystals, near-infrared (NIR) photoluminescent silver sulfide quantum dots (Ag2S QDs) have tunable optical properties and good biocompatibility (Hong et al., 2012; Yang, Zhao, Zhang, Xiong & Yu, 2013). Functionalized Ag2S QDs have been synthesized for targeted small animal imaging (Jiang, Zhu, Zhang, Tian & Pang, 2012; Tan et al., 2013), since the Ag2S QDs are regarded as excellent NIR imaging probes. Via covalence bonding or coordination, ligand-capping Ag2S QDs can be conjugated with polymeric materials to form functional nanocomposites. In our previous work (Tan, Wan & Li, 2013), glutathione (GSH)-capping Ag2S QDs were encapsulated by modified CS with the aid of ethylene diamine tetraacetic acid (EDTA). In that case, the optical properties of the Ag2S QDs were hardly influenced by the CS matrix. If Ag2S QDs capped with hydrophobic chains are incorporated with the acid-sensitive CS, we will expect pH-responsive and photoluminescent nanomaterials for loading and controlled release of hydrophobic drugs. In the present work, we present newly designed core-shell nanostructured materials, i.e., CS-encapsulating Ag2S QDs with entrapped doxorubicin (DOX) for both drug release and NIR 3

imaging.

Oleic

acid-capping

Ag2S

QDs

were

first

synthesized,

reacted

with

N-hydroxysuccinimide and conjugated with CS at the amino sites. Coexisting with DOX in aqueous solution, the hydrophobic oleoyl groups were prone to form local aggregates and trap DOX by hydrophobic interaction, resulting in Ag2S(DOX)@CS nanospheres spontaneously (Scheme 1). The oleoyl-CS chains are sensitive to environmental pH changes. At pH≥7.0, the DOX is encaged in the nanospheres due to the strong hydrophobic interaction between the oleoyl groups. At low pH, in contrast, the protonated amine groups increase the charge repulsion between the oleoyl-CS chains and lead to the release of DOX through chains expansion (Scheme 1). The NIR photoluminescent Ag2S QDs serve as fluorescent tags, by which the Ag2S (DOX)@CS nanospheres can be imaged and traced when used for drug delivery. Aiming at developing an anticancer drug delivery system with rapid response to intracellular pH as well as excellent NIR photoluminescent properties for in vivo imaging, the core-shell Ag2S(DOX)@CS nanospheres were thoroughly characterized in this paper.

2. Experimental 2.1. Materials Silver nitrate (AgNO3, 99.9 %), (C2H5)2NCS2Na·3H2O(Na(DDTC) and chitosan (CS, weight-average molecular weight Mw = 21000, degree of acetylation ≥ 95 %) were provided by Sinopharm Chemical Reagent Co., Ltd, China.1-octadecene (98%), oleic acid (99 %), N-hydroxysuccinimide (99 %), fetal bovine serum (FBS), dulbecco's modified Eagle's medium (DMEM),

3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl

tetrazolium

bromide

(MTT),

and

doxorubicin (DOX) hydrochloride (> 98 %) were purchased from Sigma-Aldrich. Millipore water was used in all experiments. All other chemicals and solvents were provided by Sinopharm Chemical Reagent Co., Ltd and used as received without further purification (analytically pure). 2.2. Synthesis of Ag2S QDs Oleic acid-capping Ag2S QDs were synthesized according to the following protocol: 0.1 mmol of (C2H5)2NCS2Ag, which was first synthesized following a reported procedure (Du et al., 2010), was added into 10 mmol of oleic acid and 25 mmol of 1-octadecene in a three-necked flask at room temperature under vigorous stirring. The mixture was heated to 60 °C in vacuum to remove residual water and oxygen, followed by being heated to 210 °C and kept at 210 °C for 1 h in 4

nitrogen atmosphere. Then the mixture was cooled down to room temperature naturally, into which successive ethanol was added. The products were collected by centrifugation at 6830 g for 20 min. Thereafter, a mixture of as-synthesized Ag2S QDs, 12 mmol of thiolated oleic acid, 30 mmol of 1-octadecene and 30 mmol of ethanol were stirred for 32 h at room temperature for ligand exchange. The oleic acid-capping Ag2S QDs were obtained by centrifugation with a centrifugal force of 20490 g for 20 min and washing with deionized water. 2.3. Preparation of Ag2S(DOX)@CS nanospheres The oleic acid-capping Ag2S QDs were mixed with 15mmol of NHS in 10 mL of anhydrous dimethyl formamide (DMF), to which 30 mmol of DCC was added slowly and the mixture was allowed to react under stirring for 24 h at room temperature in nitrogen atmosphere. Subsequently, the mixture was filtered, washed by ethyl ether thoroughly and rotation-evaporated to obtain Ag2S QDs capped by oleic acid N-hydroxysuccinimide ester. On the other hand, 0.5 g of CS was dissolved in 30 mL of aqueous acetic acid (1 wt%), and the pH of the solution was adjusted to 6.0 by slow addition of 0.1 M NaOH. The modified Ag2S QDs were dispersed in absolute ethanol, which was then added drop-wise to the CS solution at 95 °C and reacted for 36 h under stirring. The resultant solution was cooled to room temperature and precipitated by adding acetone and adjusting pH to 9.0. The precipitates were filtered, washed with acetone for three times, air-dried and redispersed in aqueous acetic acid. The degree of substitution (DS) on CS was determined by ninhydrin assay (Chiu et al., 2009). Thereafter, DOX was added into the above aqueous acetic acid solution of Ag2S@CS at room temperature. The predetermined weight ratio of DOX to Ag2S@CS was 1:5. After stirring in the open air for 10 min, the pH of the mixture was adjusted to 7.4, and the mixture was stirred for an additional 30 min. Then the mixture was dialyzed against water (pH 7.4) to remove free DOX. The Ag2S(DOX)@CS nanospheres were collected by centrifugation at 8000 rpm for 15 min, repeatedly washed with ethanol, and vacuum dried at room temperature for 3 h. The Ag2S(DOX)@CS nanospheres were dissolved in aqueous acetic acid (1 M, pH 2.4), whose UV-vis absorbance at 485 nm was measured to determine the total loading of DOX. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following equations (Yang et al., 2015): DLC = (weight of loaded drug/weight of polymer) × 100% 5

DLE = (weight of loaded drug/weight of drug in feed) × 100% 2.4. Characterization Sample morphology was observed using a JEM-2100 transmission electron microscope (TEM, JEOL, Japan) at 200 kV. The JEM-2100 transmission electron microscope was also used for energy dispersive X-ray spectroscopy (EDX). A small amount of sample solution was dropped on an amorphous carbon-coated copper grid, which was then freeze-dried in vacuum at -50 °C before observation. X-ray photoelectron spectroscopy (XPS) was conducted using an ESCALAB 250 X-ray photoelectron spectrometer (Thermo Scientific, US) with non-monochromatic Al Kα X-ray. The analyzer was operated at 20 eV pass energy with an energy step size of 0.1 eV. X-ray diffraction (XRD) pattern was recorded using a D/max-2200/PC X-ray diffractometer (Rigaku, Japan) fitted with nickel-filtered Cu Kα radiation. Fourier transform infrared spectroscopy (FTIR) was conducted on a NEXUS 670 FT-IR spectrometer (Thermo Nicolet, US). Thermogravimetry (TG) was conducted on a Q5000IR thermogravimetric analyzer (TA, US) under nitrogen flow from room temperature to 800 °C at a heating rate of 20 °C/min. Size distribution and zeta potential of samples were determined by a Nano ZS90 particle size and zeta potential analyzer (Malvern, UK) based on dynamic light scattering (DLS) at a scattering angle of 90°. Absorption spectra were recorded on a Lambda 750S UV-Vis-NIR spectrophotometer (PerkinElmer, US), background corrected for any water contribution. NIR emission spectra were collected with a Fluorolog-3 fluorescence spectrophotometer (Horiba JovinYvon, France) equipped with an external 0-10 W adjustable continuous wave laser (300-1600 nm). The focal spot size of the laser was set at 1 cm2. The Ag concentration in different parts of mice was determined on an X series inductively coupled plasma mass spectrometer (ICP-MS) (Thermo Elemental, UK). 2.5. Measurement of DOX release 2 mL of Ag2S QDs(DOX)@CS/PBS solutions (1 mg/mL) was transferred into a dialysis tube (MWCO = 1000 Da), which was then immersed in a beaker filled with 50 mL of PBS buffer at varied pH values (5, 6 and 7.4) to be measured at 37 °C. At predetermined time intervals, 5 mL of external solution was withdrawn and analyzed using a Synergy 2 Multi-Mode Reader (BioTek, US). The beaker was immediately refilled with 5 mL of fresh PBS of the same composition and pH for the next sampling. The cumulative release of DOX was determined by measuring the 6

fluorescence intensity at 580 nm under excitation of 485 nm laser. The experiments were performed in triplicate. The cumulative DOX release is calculated according to the following equation (Yang et al., 2015):

Where Er is the total cumulative release% of DOX; Ve is the replacement of PBS volume (5 mL); V0 is the total volume of PBS (50 mL); Ci is DOX concentration of the i-th replacement liquid (µg/mL) (determined by fluorescence measurement); Cn is DOX concentration of the last replacement liquid (µg/mL); mdrug is the total amount of DOX encapsulated (µg). 2.6. Cell culture HeLa cells (mouse colonic cancer cells) purchased from Institute of Biochemistry and Cell Biology, Chinese Academy of Science, were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10 wt% FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin in a humidified incubator with 5 vol% carbon dioxide at 37 °C. The medium was refreshed every 2 or 3 days according to cell density. 2.7. Cell imaging 0.5 mL of HeLa cell suspension was transferred to an eight-well Lab-Tek II chamber slide (Nalge Nunc, Napevillem, IL), followed by removing the culture medium and adding a Ag2S QDs(DOX)@CS/PBS solution (10 μg/mL). The cells were incubated with the nanospheres for 2 h to allow internalization. At predetermined time points, the medium was aspirated from the wells, and the cells were rinsed with fresh culture medium for three times. The cell fluorescence excited at 488 nm or 808 nm was observed by using a confocal laser scanning microscope (Zeiss LSM 710, Germany) or a liquid nitrogen cooled InGaAs camera (Princeton Instruments, US) with a sensitivity ranging from 800 to 1700 nm. 2.8. MTT assay Cytotoxicity of Ag2S@CS nanospheres and Ag2S(DOX)@CS nanospheres was evaluated by MTT assay. HeLa cells were seeded in 96-well culture plates at a density of 5000 cells per well and incubated at 37 °C for 24 h for cell attachment. The culture medium in each well was then replaced by a fresh medium containing Ag2S@CS nanospheres and Ag2S(DOX)@CS nanospheres 7

at varied concentrations (0.01 - 100mg/mL). One row of the 96-well plates was used as a control group. After further incubation for 24 h, the culture plates were rinsed with a PBS buffer (0.01 M, pH 7.4) to remove unattached cells and the remaining cells were treated with 5 mg/mL MTT stock solution in PBS for 4 h. The medium containing unreacted MTT was then carefully removed. The obtained formazan was dissolved in DMSO, and the absorbance of individual wells was recorded at 570 nm using a Multiskan MK3 Enzyme-labeled Instrument (Thermo Scientific, US). The blank was subtracted to the measured optical density (OD) values. The cell viability was determined by the following equation: Cell viability % = Absorbance of test cells/Absorbance of control cells × 100 % 2.9. Animal tumor model A mouse HeLa tumor model was utilized to evaluate in vivo tumor reduction by Ag2S(DOX)@CS nanospheres. Animal studies were conducted according to approved protocols by the Animal Ethics Committee of Shanghai Jiao Tong University. Seven weeks old female BALB/c mice (~19 g) were provided by Institute of Biochemistry and Cell Biology, Chinese Academy of Science. Tumor inoculation was performed by injecting ~1×105 HeLa cells subcutaneously into the mice. The cells were washed twice in PBS and re-suspended in sterile normal saline prior to injection. The tumors were allowed to grow to a volume of ~100 mm3, which was estimated by V=1/2 (l) × (w)2, where l is the longest tumor diameter and w is the shortest tumor diameter measured by a digital caliper (Camacho, et al., 2015). The HeLa tumor-bearing mice were randomly divided into experimental and control groups, and were treated with tail vein injections of PBS, DOX (0.54 mg/kg), Ag2S@CS (9.7 mg/kg) and Ag2S(DOX)@CS nanospheres (10.2 mg/kg) every other day for a total of 12 days postinoculation. Thereafter, the mice were sacrificed, and tumors were harvested and measured, based on which tumor growth inhibition was assessed. 2.10. Small animal imaging The HeLa tumor-bearing mice were anesthetized with 10 % chloral hydrate via intraperitoneal injection. Then 20 μg of Ag2S(DOX)@CS nanospheres (0.1 mg/mL in PBS) was injected into tail vein of the nude mice. After 6 h, 12 h and 24 h, the mice were positioned on an imaging platform, and NIR fluorescence images were recorded using the liquid-nitrogen-cooled InGaAs camera by collecting photons in NIR-II region. The excitation source was an 808 nm laser diode coupled to a 8

4.5 mm focal length collimator. The emission from the animal was filtered through a 900 nm long-pass filter coupled with the InGaAs camera. The excitation power density at the imaging platform was 140 mW/cm2.

3. Results and discussion 3.1. Structure and morphology The morphology of as-synthesized Ag2S QDs was observed by TEM. A TEM image in Fig.1a shows monodisperse nanoparticles that are Ag2S QDs. A high-resolution TEM image in Fig.1b indicates lattice fringes of the Ag2S QDs with an interplanar spacing of ~ 0.24 nm, which could be assigned to the (-112) facets of monoclinic α-Ag2S crystal (Zhang, Liu, Li, Chen & Wang, 2014). EDX analysis result in Fig.1c confirms the chemical composition and atomic ratio of the Ag2S QDs. The XRD pattern of the Ag2S QDs in Fig.1d shows positions and relative intensities of the diffraction peaks that match monoclinic Ag2S (JCPDS card No. 14-0072). The TEM images of Ag2S(DOX)@CS nanospheres are shown in Fig. 2a and Fig. 2b. The nanospheres in the image had spherical outline and a narrow size distribution (Fig. 2a). The image with a larger magnification indicates the absence of free standing Ag2S QDs outside the nanospheres (Fig. 2b). TG analysis can be used for determining the loading of Ag2S QDs in the Ag2S(DOX)@CS nanospheres, as the organic shell decomposes and volatilizes at high temperature and the Ag2S QDs are left. As shown in Fig.2c, the mass of nanospheres gradually decreased with increasing temperature .The TG curve became a flat line at beyond 650 °C and the remaining mass was 10.08 % of the total. Therefore, loading of Ag2S QDs in the nanospheres was 10.08 wt%. The size distribution of the nanospheres determined by DLS is shown in Fig. 2d. The average diameter was 33.4 nm with a polydispersity index (PDI) of 0.116. FTIR spectroscopy was adopted to evidence the conjugation of the oleic acid-capping Ag2S QDs with CS. As demonstrated in Fig. 2e, the FTIR spectrum of the Ag2S@CS showed stronger absorption peaks at 1664 cm-1 and 1572 cm-1 compared with the CS, assigned to C=O stretching vibration (amide I band) and N–H bending vibration (amide II band) of amide groups, respectively. In addition, the absorption intensity at 2850-2932 cm-1characteristic of –(CH2)– for the Ag2S@CS increased significantly in comparison with the CS. The FTIR results suggest that the Ag2S QDs were conjugated with the CS successfully. 9

3.2. Optical properties The optical absorption and emission properties of the Ag2S(DOX)@CS nanospheres in NIR region were investigated. Fig. 3a shows the vis-NIR absorption spectra of Ag2S QDs and Ag2S(DOX)@CS nanospheres dispersed in aqueous acetic acid. The Ag2S QDs exhibited an absorption in the NIR region at ~840 nm. For the Ag2S(DOX)@CS nanospheres, the absorption was found at nearly the same wavelength. The NIR emission spectra of Ag2S QDs and Ag2S(DOX)@CS nanospheres excited at 808 nm are shown in Fig. 3b. An emission peak centered at ~1032 nm with a full width at half maximum (FWHM) of 41 nm was observed for bothAg2S QDs and Ag2S(DOX)@CS nanospheres, and the emission intensities of the two samples were similar. These results indicate that the optical properties of the Ag2S QDs were hardly influenced by the conjugation with CS. Fluorescence quantum yield (QY) of the Ag2S(DOX)@CS nanospheres was measured using indocyanine green (ICG) as a reference (QY = 13% in DMSO). The QY of the Ag2S(DOX)@CS nanospheres was calculated following the equation (Tan, Wan & Li, 2013):

NS

 F  A  n   ICG   NS  *  ICG  *  NS   FICG   ANS   nICG 

2

Where  NS , FNS , ANS and nNS are the QY, integrated fluorescence intensity, integrated absorption and refractive index of the solvent for the Ag2S(DOX)@CS nanospheres. The parameters with a subscript of ICG are corresponding quantities of the solvent for ICG. The Ag2S(DOX)@CS nanospheres exhibited a QY of 12.4±1.0%, which is qualified for NIR imaging in vivo.

3.3. pH-triggered drug release The DS of Ag2S(DOX)@CS nanospheres may exert significant influences on their structure and drug loading capacity. We prepared Ag2S(DOX)@CS nanospheres with different DSs and measured the diameter, zeta potential, DLC and DLE of these samples at pH 7.4 and 5.0. As listed in Table 1, both DLC and DLE increased with the increase of DS, since a larger DS means a greater portion of hydrophobic chain moiety where more drugs can be entrapped. For all the 10

samples, the diameter and zeta potential at pH 5.0 were larger than those at pH 7.4, indicating the acid responsiveness of the nanospheres. It is noticeable that, however, the differences in diameter and zeta potential of the sample with DS=15 % between at pH 5.0 and at pH 7.4 were much smaller than those of the other samples. Although this sample had high DLC and DLE, its sensitivity to the environmental pH became poor due to a strong association of the oleoyl-CS chains, and the drug delivery capability of the nanospheres deteriorated accordingly. Therefore, the Ag2S(DOX)@CS nanospheres with a DS of 12 % were chosen for the study.

Influences of pH changes on the size and zeta potential of Ag2S(DOX)@CS nanospheres with the DS of 12 % were depicted in Fig. 4a. At low pH values, the amine groups on the CS shell were strongly protonated, as indicated by the high zeta potential. The charge repulsion between protonated amine groups dominated, resulting in the extension of polymer chains and thus larger size of the nanospheres. With increasing pH, the electrostatic repulsion between the CS polymeric chains was reduced due to the deprotonation of the amine groups (lowered zeta potential). On this occasion, the hydrophobic interaction between the oleoyl chains dominated, leading to a gradual reduction in the size of the nanospheres. This pH-triggered transition from a highly charged stretching structure to a weakly charged contracting structure enables the Ag2S(DOX)@CS nanospheres to act as an on-off switch in response to the changes in ambient pH. The cumulative amount of DOX released from the Ag2S(DOX)@CS nanospheres at different pH values was measured by identifying the fluorescence signal typical of DOX. The release profiles over a 30 h period are shown in Fig. 4b. Most of the DOX molecules were entrapped in the compact nanospheres at pH 7.4, and only a small dose of DOX was released. At lower pH values, the nanospheres became swollen because of stretched molecular chains of the shell, and more DOX was thus released. As a control, the release profile of a DOX solution at pH 5.0 was shown to demonstrate the release media was appropriate. The pH in endosome and lysosome of cancer cells usually lies in the range of 4-6. The acidic intracellular environment can trigger DOX release from the Ag2S(DOX)@CS nanospheres.

3.4. In vitro and in vivo drug release and bioimaging In vitro cell imaging was performed to investigate the intracellular drug release and NIR 11

emission from the Ag2S(DOX)@CS nanospheres. HeLa cells were incubated with the Ag2S(DOX)@CS nanospheres for 12 h to allow internalization of the nanospheres and the subsequent release of DOX in the cells. Then the cells were observed under excitation at visible-light and NIR-I wavelengths, respectively. After a long-period incubation, the intracellular Ag2S(DOX)@CS nanospheres entered the endolysosomal system (Xu et al., 2015) and responded to lowered pH. DOX was released from the swollen nanospheres, as evidenced by readily detectable red fluorescence from the cells under irradiation of 488 nm laser (Fig. 5a). Excited at 808 nm, NIR fluorescence originating from the Ag2S QDs was observed (Fig. 5b), indicating the cell imaging ability of the Ag2S(DOX)@CS nanospheres. Different from the image in Fig. 5a where the DOX molecules were distributed throughout the cells, the NIR signal showed punctuated cytoplasmic distribution, indicating the nanospheres were mainly located in the cytoplasm. MTT assay was conducted for HeLa cells containing Ag2S@CS nanospheres or Ag2S(DOX)@CS nanospheres to assess the cytotoxicity of the carrier materials and the cancer cell killing ability of the drug-loading nanospheres. As shown in Fig. 5c, the cell viability changed slightly with increasing concentration of theAg2S@CS nanospheres after incubation for both 12 and 24 h. At a high concentration of 1 mg/mL, more than 85 % of the cells survived 24 h incubation, indicating low toxicity of the carrier materials to the cells. In the case of Ag2S(DOX)@CS nanospheres, the cell viability decreased significantly with the increase of concentration, and a longer incubation time led to even more dead cells. After incubation with 1 mg/mL Ag2S(DOX)@CS nanospheres for 24 h, only 22.2 % of the cells were living. The intracellular early/late endosomes and lysosomes have a pH of 5-6.5 and 4.5-5.5, respectively. This pH gradient could prevent premature release of DOX from the nanospheres and help enhance the drug dose acting on the nucleus. IC50 (a concentration at which 50 % of the cells survive) of the Ag2S(DOX)@CS nanospheres determined by the MTT assay was 90.2±1.3 μg/mL. At the same equivalent DOX concentration of 50 μg/mL, the Ag2S(DOX)@CS nanospheres exhibited much greater cancer cell inhibition than free DOX (Fig. 5e). All these results indicate that the Ag2S(DOX)@CS nanospheres can release DOX in response to intracellular pH and kill the cancer cells efficiently.

12

To further investigate the antitumor efficacy of Ag2S(DOX)@CS nanospheres, free DOX, Ag2S@CS nanospheres, Ag2S(DOX)@CS nanospheres or PBS as a control was intravenously injected into BALB/c mice bearing a HeLa tumor via tail vein every other day. The tumor volume and body weight of the treated mice were monitored over 12 days. As shown in Fig. 6a, the average tumor volume of the mice treated with Ag2S@CS nanospheres was close to that of the mice treated with PBS at 12 days postinjection. For the mice treated with the Ag2S(DOX)@CS nanospheres, the tumor growth was greatly inhibited over the same period, as reflected by a 48.5 % reduction in tumor volume compared with the control. The Ag2S(DOX)@CS nanospheres were delivered to the tumor site under the effect of enhanced permeability and retention (EPR) and were then internalized by the tumor cells. DOX release was triggered when the nanospheres entered the cellular compartments such as early/late endosomes and lysosomes. The sensitivity of the nanospheres to pH changes facilitates rapid escape of the nanospheres from the endolysosomal system and reduces the multiple drug resistance (MDR) effect (Xu et al., 2015). As can be seen in Fig. 6b, the mice treated differently showed only slight changes in body weight after the 12-day period, indicating that the Ag2S(DOX)@CS nanospheres were well-tolerated without severe side effects. Distribution of the element Ag in the tumor-bearing mice at different time intervals after injection of Ag2S(DOX)@CS nanospheres was examined using ICP-MS technique (Fig. 6c). The Ag concentration in the tumor was much higher than those in five major organs, demonstrating that the nanospheres tended to accumulate in the tumor attributed to the EPR effect. The Ag concentration in liver and spleen was higher compared to that in stomach, heart or kidney, revealing a relatively high affinity of the nanospheres for liver and spleen. In addition, the Ag concentration in the tumor and the organs began to decrease after 6 h due to metabolism. Compared with those in the organs, the downward trend of Ag level in the tumor was less obvious, indicating longer retention of the nanospheres inside the tumor. To visualize the distribution of Ag2S(DOX)@CS nanospheres in a living body when used for therapeutic purposes, noninvasive in vivo NIR imaging of nude mice was carried out. The nanospheres were dispersed in PBS, which were administered into anesthetized nude mice by tail vein injection. At different time intervals post-injection, the mice were imaged under 808 nm excitation. The NIR fluorescence derived from the nanospheres in the mice was readily detected 13

for real-time monitoring, as shown in Fig. 6c. At 6 h post-injection, the nanospheres were distributed in the mice body via blood circulation (Fig. 6c (i)). It is noted that the fluorescence from the tumor was stronger than that from other regions of the mice body, such as liver, spleen, heart, etc. At 12 h post-injection, the fluorescence intensity became still higher, indicating that the nanospheres accumulated in the tumor by the EPR effect (Fig. 6c (ii)). The fluorescence signals from the mice body were still observable after 24 h with reduced intensity (Fig. 6c (iii)), attributed to clearance of the nanospheres by metabolism. However, the fluorescence intensity still maintained at a high level at the tumor site, revealing that the nanospheres could stay in the tumor for a long time. Moreover, a tumor was harvested from a sacrificed mouse at 24 h post-injection and was imaged (Fig. 6c (iv)). The five major organs of the same mouse were also harvested for comparison (Fig. 6c (v)). The in and ex vivo imaging results demonstrate that the Ag2S(DOX)@CS nanospheres can accumulate in the tumor after being injected into a mouse body and release the anticancer drug under intracellular environment for tumor inhibition.

4. Conclusions In summary, we have demonstrated synthesis and evaluations of core-shell structured Ag2S(DOX)@CS nanospheres capable of releasing DOX under acidic environment and emitting NIR-II fluorescence for bioimaging under NIR-I irradiation. By conjugating the oleic acid-capping Ag2S QDs with pH-sensitive CS at an appropriate DS, CS-based bifunctional nanospheres with good biocompatibility were successfully prepared. The oleoyl-CS chains could entrap DOX within the hydrophobic oleoyl groups via hydrophobic interaction and extended at lowered pH to release the otherwise encaged DOX. The prompt response to ambient pH changes enabled rapid escape of the nanospheres from the endolysosomal system to target nuclei, which enhanced antitumor efficacy of the released DOX. Besides, the encapsulated Ag2S QDs could emit bright NIR fluorescence qualified for in vitro and in vivo imaging, by which the distribution of the nanospheres in a body can be traced. This study gives an insight in to developing CS-based nanocomposite drug carriers for efficient cancer therapy.

Acknowledgements This work was financially supported by the National Natural Science Foundation (Grant No. 14

51403125, 81671802 and 11505110) and the Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20130073120087).

References Camacho, K. M., Kumar, S., Menegatti, S., Vogus, D. R., Anselmo, A. C., Mitragotri, S. (2015). Synergistic antitumor activity of camptothecin-doxorubicin combinations and their conjugates with hyaluronic acid. Journal Controlled Release, 210, 198-207. Chen, K-J., Chiu, Y-L., Chen, Y-M., Ho, Y-C. & Sung, H-W. (2011). Intracellularly monitoring/imaging the release of doxorubicin from pH-responsive nanoparticles using Förster resonance energy transfer. Biomaterials, 32, 2586-2592. Chiu, Y. L., Chen, S. C., Su, C. J. Hsiao, C. W., Chen, Y. M., Chen, H. L. & Sung, H. W. (2009). pH-triggered injectable hydrogels prepared from aqueous N-palmitoyl chitosan: in vitro characteristics and in vivo biocompatibility. Biomaterials, 30, 4877-4888. Du, Y. P., Xu, B., Fu, T., Cai, M., Li, F., Zhang, Y. & Wang, Q. B. (2010). Near-Infrared Photoluminescent Ag2S Quantum Dots from a Single Source Precursor. Journal of American Chemistry Society, 132, 1470-1471. Ferlay, J., Steliarova-Foucher, E., Lortet-Tieulent, J., Rosso, S., Coebergh, J. W. W., Comber, H., Forman, D. & Bray, F. (2013). Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. The European Journal of Cancer, 49, 1374-1403. Flitney, F. W., Megson, I. L., Thomson, J. L. M., Kennovin, G. D. & Butler, A. R. (1996) Vasodilator responses of rat isolated tail artery enhanced by oxygen-dependent, photochemical release of nitric oxide from iron-sulphur-nitrosyls. Britain Journal of Clinical Pharmacology, 117, 1549-1557. Geszke-Moritz, M. & Moritz, M. (2013). Quantum dots as versatile probes in medical sciences: synthesis, modification and properties. Materials Science and Engineering C, 33, 1008-1021. Hong, G., Robinson, J., Zhang, Y., Diao, S., Antaris, A. L. & Wang, Q. (2012). In vivo fluorescence imaging with Ag2S quantum dots in the second near-infrared region. Angewandte Chemie International Edition, 51, 9818-9821.

15

Jayasree, S., Sasidharan, A., Koyakutty, M., Nair, S., Menon, D. (2011). Mannosylated chitosan-zinc sulphide nanocrystals as fluorescent bioprobes for targeted cancer imaging. Carbohydrate Polymers, 85, 37-43. Jiang, P., Zhu, C. N., Zhang, Z. L., Tian, Z. Q. & Pang, D. W. (2012) Water-soluble Ag2S quantum dots for near-infrared fluorescence imaging in vivo. Biomaterials, 33, 5130-5135. Li, M., Tang, Z., Sun, H., Ding, J., Song, W., & Chen X. (2013). pH and reduction dual-responsive nanogel cross-linked by quaternization reaction for enhanced cellular internalization and intracellular drug delivery. Polymer Chemistry, 4, 1199-1207. Li, S-Y., Sun, R., Wang, H-X., Shen, S., Liu, Y., Du, X-J., Zhu, Y-H. & Wang, J. (2015). Combination therapy with epigenetic-targeted and chemotherapeutic drugs delivered by nanoparticles to enhance the chemotherapy response and overcome resistance by breast cancer stem cells. Journal Controlled Release, 205, 7-14. Linderoth, L., Fristrup, P., Hansen, M., Melander, F., Madsen, R., Andresen, T. L. & Peters, G. H. (2009). Mechanistic study of the sPLA2-mediated hydrolysis of a thio-ester pro anticancer ether lipid. Journal of American Chemistry Society, 131, 12193-12200. Lv, S., Tang, Z., Zhang, D., Song, W., Li, M., Lina, J., Liu, H. & Chen, X. (2014). Well-defined polymer-drug conjugate engineered with redox and pH-sensitive release mechanism for efficient delivery of paclitaxel. Journal Controlled Release, 194, 220-227. Montembault, A., Viton, C. & Domard, A. (2005). Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent. Biomacromolecules, 6, 653-662. Qiao, Z. Y., Qiao, S. L., Fan, G., Fan, Y. S., Chen, Y. & Wang, H. (2014). One-pot synthesis of pH-sensitive poly (RGD-co-β-amino ester) s for targeted intracellular drug delivery. Polymer Chemistry, 5, 844-853. Rinaudo, M. Chitin and chitosan: Properties and applications. (2006). Progress in Polymer Science, 31, 603-632. Tan, L., Wan, A., Li, H. & Lu, Q. (2012). Novel quantum dots-carboxymethyl chitosan nanocomposite nitric oxide donors capable of detecting release of nitric oxide in situ. Acta Biomateriallia, 8, 3744-3753. Tan, L., Wan, A. & Li, H. (2013). Conjugating S-nitrosothiols with glutathiose stabilized Ag2S quantum dots for controlled nitric oxide release and near-infrared fluorescence imaging. ACS 16

Applied Materials and Interfaces, 5, 11163-11171. Tan, L., Wan, A. & Li, H. (2013). Ag2S quantum dots conjugated chitosan nanospheres towards light-triggered nitric oxide release and near-infrared fluorescence imaging. Langmuir, 29, 15032-15042. Wang, D., Su, Y., Jin, C., Zhu, B., Pang, Y., Zhu, L., Liu, J., Tu, C., Yan, D. & Zhu, X. (2011), Supramolecular Copolymer Micelles Based on the Complementary Multiple Hydrogen Bonds of Nucleobases for Drug Delivery. Biomacromolecules, 12, 1370-1379. Xu, H., Yang, D., Cai, C., Gou, J., Zhang, Y., Wang, L., Zhong, H. & Tang, X. (2015). Dual-responsive mPEG-PLGA-PGlu hybrid-core nanoparticles with a high drug loading to reverse the multidrug resistance of breast cancer: An in vitro and in vivo evaluation. Acta Biomateriallia, 16, 156-168. Yang, H. Y., Zhao, Y. W., Zhang, Z. Y., Xiong, H. M. & Yu, S. N. (2013). One-pot synthesis of water-dispersible Ag2S quantum dots with bright fluorescent emission in the second near-infrared window. Nanotechnology, 24, 055706. Yang, Q., He, C., Xu, Y., Liu, B., Shao, Z., Zhu, Z., Hou, Y., Gong, B. & Shen, Yu-M. (2015). Chitosan oligosaccharide copolymer micelles with double disulphide linkage in the backbone associated by H-bonding duplexes for targeted intracellular drug delivery. Polymer Chemistry, 6, 1454-1464. Yang, Q., Tan, L., He, C., Liu, B., Xu, Y., Zhu, Z., Shao, Z., Gong, B. & Shen, Y-M. (2015). Redox-responsive micelles self-assembled from dynamic covalent block copolymers for intracellular drug delivery. Acta Biomaterialia, 17, 193-200. Yang, X. Z., Dou, S., Sun, T. M., Mao, C. Q., Wang, H. X. & Wang, J. (2011). Systemic delivery of siRNA with cationic lipid assisted PEG-PLA nanoparticles for cancer therapy. Journal Controlled Release, 156, 203-211. Zhang, Y., Liu, Y., Li, C., Chen, X. & Wang, Q. (2014). Controlled Synthesis of Ag2S Quantum Dots and Experimental Determination of the Exciton Bohr Radius. The Journal of Physical Chemistry C, 118, 4918-4923. Zhou, Y. F., Huang, W., Liu, J. Y., Zhu, X. Y. & Yan, D. Y. (2010). Self-Assembly of Hyperbranched Polymers and Its Biomedical Applications. Advanced Materials, 22, 4567-4590. Hubbell, J. A. & Chilkoti, A. (2012) Nanomaterials for drug delivery. Science, 337, 303-305. 17

Fig. 1.(a) TEM image and (b) high-resolution TEM image of as-synthesized Ag2S QDs. (c) EDX spectrum of the Ag2S QDs. (d) Wide-angle XRD pattern of the Ag2S QDs.

Fig. 2. (a) TEM image and (b) high-resolution TEM image of Ag2S(DOX)@CS nanospheres. (c) TG curve of Ag2S(DOX)@CS nanospheres. (d) Size distribution of Ag2S(DOX)@CS nanospheres at pH 7.4. (e) FTIR spectra of CS and Ag2S@CS.

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Fig. 3. (a) Vis-NIR absorption spectra and (b)NIR PL spectra of Ag2S QDs and Ag2S(DOX)@CS nanospheres.

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Fig. 4. (a) Diameter and zeta potential of Ag2S(DOX)@CS nanospheres as a function of pH value. The data are presented as average ± standard deviation (n=3). (b) DOX release from Ag2S(DOX)@CS nanospheres in PBS buffers of different pH values at 37 °C. The data are presented as average ±standard deviation (n=3).

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Fig. 5. (a) Fluorescence image excited at 488 nm and (b) NIR image excited at 808 nm of HeLa cells incubated with Ag2S(DOX)@CS nanospheres for 12 h. The fluorescence image was acquired in a wavelength window between 560-600 nm (DOX channel). The NIR image was acquired by a InGaAs camera sensitive to signals in NIR region and expressed by pseudocolor in an image. The scale bar in the images represents 25 μm. (c) Viability of HeLa cells incubated with different concentrations of (c) Ag2S@CS nanospheres and (d) Ag2S(DOX)@CS nanospheres for 12 and 24 h, respectively. The data are relative to the control. Statistical significance: *P < 0.05. (e) Cytotoxicity of free DOX and Ag2S(DOX)@CS nanospheres to HeLa cells at an equivalent DOX concentration of 50 μg/mL after incubation for 24 h. Statistical significance: *P < 0.05. All the cell 21

viability data are presented as average ± standard deviation (n = 5).

22

Fig. 6. (a) Average volume of mice-bearing HeLa tumors at different time points after intravenous injection of PBS, free DOX, Ag2S@CS and Ag2S(DOX)@CS nanospheres via tail vein. Statistical significance: *P < 0.05. (b) Body weight changes of the tumor-born mice after treatment with PBS, free DOX, Ag2S@CS and Ag2S(DOX)@CS nanospheres. The data are presented as average ± standard deviation (n = 3). (c) ICP-MS analysis of tumor and five major organs of the mice sacrificed at different time points after injection of Ag2S(DOX)@CS. The data are presented as average ± standard deviation (n = 5). Statistical significance: *P < 0.05. (d) In vivo NIR images of a nude mouse at 6 h (i), 12 h (ii) and 24 h (iii) after injection of the Ag2S(DOX)@CS nanospheres; ex vivo NIR image of the tumor (iv) and the organs (v) harvested from the sacrificed nude mouse.

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Scheme 1. Schematic synthesis procedure of Ag2S(DOX)@CS nanospheres and mechanisms of pH-triggered DOX release and NIR imaging.

24

Table 1 Influences of degree of substitution on diameter, zeta potential, DOX loading content and loading efficiency of the Ag2S(DOX)@CS nanospheres.

DS (%)

Diameter at pH 7.4

Diameter at pH

Zeta potential

Zeta potential

DLC (%)

DLE (%)

(nm)

5.0 (nm)

at pH 7.4 (mV)

at pH 5.0 (mV)

5

45.9±3.3

101.3±4.1

5.33±1.47

54.7±3.8

3.01±0.4

13.5±1.1

8

44.1±53.7

92.0±4.7

5.25±1.67

50.9±3.5

3.63±0.3

26.0±1.9

10

40.6±2.7

80.8±4.1

5.13±1.73

46.1±3.4

4.32±0.4

32.4±1.8

12

37.4±3.4

71.9±3.8

5.21±1.30

37.3±3.1

4.93±0.5

36.7±2.5

15

35.5±3.6

48.5±4.2

5.15±1.67

31.5±3.0

5.87±0.7

38.3±2.3

25