Dual-stimuli responsive hyaluronic acid-conjugated mesoporous silica for targeted delivery to CD44-overexpressing cancer cells

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Dual-stimuli responsive hyaluronic acid-conjugated mesoporous silica for targeted delivery to CD44-overexpressing cancer cells Qinfu Zhao a, Jia Liu a, Wenquan Zhu b, Changshan Sun a, Donghua Di a, Ying Zhang c, Pu Wang d, Zhanyou Wang d, Siling Wang a,⇑ a

Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning Province 110016, China Institute of Medicine, Qiqihar Medical University, Qiqihar 161006, China School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, China d College of Life and Health Sciences, Northeastern University, Shenyang 110004, China b c

a r t i c l e

i n f o

Article history: Received 6 January 2015 Received in revised form 10 May 2015 Accepted 11 May 2015 Available online xxxx Keywords: Mesoporous silica nanoparticles Dual-stimuli responsive Doxorubicin Hyaluronic acid Disulfide bonds

a b s t r a c t In this paper, a redox and enzyme dual-stimuli responsive delivery system (MSN-SS-HA) based on mesoporous silica nanoparticles (MSN) for targeted drug delivery has been developed, in which hyaluronic acid (HA) was conjugated on the surface of silica by cleavable disulfide (SS) bonds. HA possesses many attractive features, including acting as a targeting ligand and simultaneously a capping agent to achieve targeted and controlled drug release, prolonging the blood circulation time, and increasing the physiological stability and biocompatibility of MSN. The anticancer drug doxorubicin (DOX) was chosen as a model drug. In vitro drug release profiles showed that the release of DOX was markedly restricted in pH 7.4 and pH 5.0 phosphate buffer solution (PBS), while it was significantly accelerated upon the addition of glutathione (GSH)/hyaluronidases (HAase). In addition, the release was further accelerated in the presence of both GSH and HAase. Confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS) showed that MSN-SS-HA exhibited a higher cellular uptake via cluster of differentiation antigen-44 (CD44) receptor-mediated endocytosis compared with thiol (SH)-functionalized MSN (MSN-SH) in CD44 receptor over-expressed in human HCT-116 cells. The DOX-loaded MSN-SS-HA was more cytotoxic against HCT-116 cells than NIH-3T3 (CD44 receptor-negative) cells due to the enhanced cellular uptake of MSN-SS-HA. This paper describes the development of an effective method for using a single substance as multi-functional material for MSN to simultaneously regulate drug release and achieve targeted delivery. Ó 2015 Published by Elsevier Ltd.

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1. Introduction

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Over recent decades, great efforts have been devoted to develop targeted drug delivery systems (DDS) for selective delivery of anticancer drugs to tumors. Although some progress has been made, targeted delivery still suffers from some limitations. One major limitation is the premature drug release of the toxic drugs before reaching the target site. Maximum therapeutic efficacy can be realized by using stimuli-responsive targeted DDS that can effectively reach specific target sites/cells without drug leakage on the way, be internalized by endocytosis via specific interactions with receptors on the tumor cells, and then rapidly release loaded drugs according to the environmental stimuli. Hence, stimuli-responsive release

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⇑ Corresponding author. Tel./fax: +86 24 23986348. E-mail address: [email protected] (S. Wang).

and active targeting are keys to increase the therapeutic efficacy of anticancer drugs. Recently, various ‘intelligent’ DDS that release drugs in response to internal or external stimuli, such as changes in pH [1–3], redox potential [3–5], enzymes [6], temperature and light [7], have received widespread attention. Among these stimuli, redox as an internal stimulus is especially appealing due to the significant difference in the concentration of glutathione (GSH) between extracellular fluids (ca. 10 lM) and intracellular fluids (1–10 mM) [8,9]. Moreover, the cytosolic GSH concentration in most tumor cells was at least 3-fold higher than that of normal cells [10]. Hence, the advantage of the disulfide bond is its stability in extracellular fluids and easy breakdown in intracellular fluids. Though the redox-responsive DDS have improved drug release in vitro and/or in vivo to some extent, recently, in order to further realize optimal drug release and delivery performances, several dual-stimuli responsive DDS that respond to two stimuli have been

http://dx.doi.org/10.1016/j.actbio.2015.05.010 1742-7061/Ó 2015 Published by Elsevier Ltd.

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developed [11,12]. These dual-stimuli responsive nanoparticles have unprecedented control over drug release and delivery to increase anti-cancer efficacy in vitro and/or in vivo [11]. Among various drug nanocarriers explored, mesoporous (pore diameter 2–50 nm) silica nanoparticles (MSN) have shown great potential as a stimuli-responsive vehicle due to their high surface area, size-adjustable pore size, well-defined pore structures, excellent biocompatibility and, especially, their easily functionalized surface [13]. For MSN, the easily functionalized surface facilitates simultaneous assembly of different moieties, e.g. the targeting group and different capping groups, on the same nanoparticles. However, it is difficult to assemble several different functional groups with effective concentrations on limited surface of functionalized MSN. To overcome the limited surface sites, we have employed a single substance grafted onto the nanoparticles to realize different functions. In this work, hyaluronic acid (HA) was selected as a multi-functional component to develop an ‘intelligent’ nanocarrier based on MSN. HA, also called hyaluronan, is an anionic, biodegradable, naturally occurring polysaccharide and a major extracellular matrix of connective tissues [14,15]. HA was chosen for several reasons including: (1) HA is biocompatible and its large molecular size is sufficient to hinder drug release; (2) HA can be readily degraded into low molecular weight fragments by hyaluronidases (HAase) to realize the enzyme-responsive release after being endocytosed by cancer cells [14,16], and levels of HAase are raised in several malignant tumors [17]; (3) HA is a targeting moiety owing to the specific interaction with CD44 receptors [15,18], a receptor that is overexpressed on various tumor cells, thereby enhancing the intracellular uptake of MSN and improving therapeutic efficacy [16]; (4) hydrophilic macromolecular HA has been recently used as a safer substitute for polyethylene glycol (PEG) grating to prolong the blood circulation time of nanoparticles [19,20]; (5) HA can increase the physiological stability and biocompatibility of MSN when administered intravenously [21]. In the present study, for the first time, MSN-SS-HA nanoparticles were developed and found to have great potential for cancer cell-targeted delivery and dual-stimuli responsive drug release in

response to GSH and HAase at tumor sites, where the HA shell was grafted onto the surface of MSN via cleavable disulfide bonds as described in Fig. 1. The anticancer drug doxorubicin (DOX) was used as a model drug. The CD44 receptor-mediated cellular uptake was evaluated in HCT-116 (receptor-positive) cells and NIH-3T3 (receptor-negative) cells. Moreover, the in vitro cytotoxicity of DOX-loaded MSN-SS-HA nanoparticles was evaluated.

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2. Materials and experimental conditions

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2.1. Materials

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Fluorescein isothiocyanate (FITC), 3-mercaptopropyltrimethoxy silane (MPTMS, 95%), cysteamine hydrochloride, 2,2’-Dipyridyl disulfide, glutathione (GSH), N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), sodium hyaluronic acid (molecular weight ca. 200 kDa), bovine serum albumin (BSA), hyaluronidase (HAase, P300 U/mg) and doxorubicin hydrochloride (DOX) were obtained from Sigma– Aldrich Chemical (St. Louis, MO, USA). Tetraethoxysilane (TEOS) and cetyltrimethylammonium bromide (CTAB) were obtained from Tianjin Bodi Chemical Holding Co., Ltd (Tianjin, China). Cell culture medium dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS) and penicillin–streptomycin, were obtained from GIBCO, Invitrogen Co. (Carlsbad, NM, USA). 3-(4,5-Dimethylthiazo l-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was supplied by Amreso (Solon, OH, USA). Fluorescent Hoechst 33258 was obtained from Molecular Probes Inc. (Eugene, OR, USA). All other chemicals were of analytical grade and used without further purification.

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2.2. Preparation of MSN-SH

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The MSN nanoparticles were synthesized according to the published method [2]. 1 g CTAB was first dissolved in 480 mL deionized water under vigorous stirring, and then 3.5 mL 2 M NaOH (aq) was added. After the mixture was heated to 80 °C for 30 min, 5.0 mL TEOS was added dropwise to the CTAB solution. Then, the reaction solution was vigorously stirred for 2 h and,

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Fig. 1. Schematic structure of MSN-SS-HA and dual-stimuli responsive targeted drug delivery. (A) Drug loading process of MSN-SS-HA, (B) magnified image of pore structure after the grafting of HA, (C) cell uptake through a CD44 receptor-mediated interaction, and (D) GSH and HAase triggered drug release inside the tumor cell.

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finally, the resulting solids were centrifuged, and washed with ethanol three times. The introduction of thiol using the post-grafting method was conducted by dispersing as-prepared MSN nanoparticles in 50 mL ethanol, followed by the addition of MPTMS (0.7 mL). The mixture was then refluxed at 80 °C under N2 atmosphere for 12 h. The thiol functionalized MSN (MSN-SH) was collected by centrifugation and washed with ethanol three times. To remove the template CTAB, the products were refluxed twice in a solution of 18 mL HCl (37%) and 320 mL methanol for 12 h, and then the surfactant-removed samples were centrifuged, washed and dried under vacuum for 12 h.

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2.3. Synthesis of MSN-SS-NH2 nanoparticles

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S-(2-Aminoethylthio)-2-thiopyridine Hydrochloride (Py-SS-NH2) was synthesized according to a published report [22], and its preparation was also described in the supporting information. The introduction of a disulfide bond and terminal ammonium was realized by suspending MSN-SH (400 mg) in ethanol (25 mL), followed by the addition of Py-SS-NH2 (200 mg). The mixture was stirred at room temperature for 36 h. And the product was collected by centrifugation, washed thoroughly with ethanol, and dried in vacuo at 80 °C for 12 h.

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2.4. Drug loading and the stimuli-responsive drug release

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40 mg DOX was dissolved in 8 mL distilled water and 8 mL pH 7.4 PBS. Then, MSN-SH (160 mg) was added to the mixture, sonicated for 1 h, and stirred for 24 h at 25 °C. The DOX-loaded MSN-SH is referred to as MSN-SH/DOX. The nanoparticles were centrifuged and washed thoroughly with pH 5.0 PBS to remove the adsorbed DOX molecules. Subsequently, the DOX-loaded nanoparticles were dispersed in pH 7.4 PBS, and 150 mg Py-SS-NH2 was added to the solution and stirred for a further 48 h. The mixture was centrifuged and washed with pH 7.4 PBS, and the resulting product was DOX-loaded MSN-SS-NH2. Finally, 50 mg HA was hydrated in pH 7.4 PBS overnight and then activated using EDC and NHS for 1 h. Subsequently, DOX-loaded MSN-SS-NH2 was added dropwise to the activated HA solution. The reaction mixture was stirred for 24 h. The DOX-loaded MSN-SS-HA nanoparticles are referred to as MSN-SS-HA/DOX. In order to evaluate the impact of different drug loading methods on the drug loading efficiencies, MSN-SS-NH2 nanoparticles were also used as the initial drug carrier to load DOX, and the rest of the process remained the same. The DOX-loaded sample is referred to as MSN-SS-HA/DOX-2.

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To investigate the redox and enzyme dual-stimuli responsive release, ca. 15 mg MSN-SS-HA/DOX sample and MSN-SH/DOX were dispersed in 20 mL pH 5.0 PBS and pH 7.4 PBS under different conditions, (1) 0 mM GSH and 0 UmL 1 HAase, (2) 10 mM GSH and 0 UmL 1 HAase, (3) 0 mM GSH and 150 UmL 1 HAase, and (4) 10 mM GSH and 150 UmL 1 HAase, shaking at 135 rpm (37 °C). The use of the concentration of HAase (150 UmL 1) was according to the research paper [21]. At specified time intervals, 3 mL samples of release solution were taken out and replaced with an equal volume of fresh fluid. The release of DOX from the buffer solution was monitored at 490 nm after centrifugation at 5000 rpm for 5 min.

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2.5. Degradation of HA by HAase

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To demonstrate the degradation of HA on the surface of silica by HAase, the change in the zeta potential of the nanoparticles in the presence of HAase was investigated in pH 5.0 and pH 7.4 PBS. MSN-SS-HA samples (5 mg) were dispersed in 6 mL PBS, and then 3 mg HAase was added to the mixture in a shaker maintained at 37 °C. The final concentration of HAase is 0.5 mg/mL (150 UmL 1). After a predetermined incubation time (0.5, 1, 2, 4, 8, 24 h), the samples were centrifuged, washed with PBS and then measured using a Zeta Potential Analyzer.

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2.6. FITC labeled MSN-SH and MSN-SS-HA

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FITC was covalently linked to APTMS by dispersing 2 mg FITC and 20 lL APTMS in 2 mL ethanol in the dark and stirring for 12 h. Then, 200 mg MSN-SH was dispersed in 20 mL ethanol, and mixed with 2 mL FITC/APTMS ethanol solution (1 mg/mL). After stirring under dark conditions for 24 h, the nanoparticles were centrifuged and washed with ethanol until the supernatants were colorless. The FITC labeled MSN-SS-HA was prepared by a two-step reaction. Firstly, FITC labeled MSN-SH reacted with Py-SS-NH2, and then HA was grafted onto the surface of MSN by an amidation reaction.

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2.7. Cell culture

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HCT-116 and NIH-3T3 cells were maintained in DMEM supplemented with FBS (10%), penicillin (1%), L-glutamine (1%), and streptomycin (1%) in 5% CO2 at 37 °C. The medium was routinely changed every 2 days and the cells were separated by trypsinisation before reaching confluence. The cell doubling times for the two cell lines were about 3 days.

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Fig. 2. SEM (A) and TEM (B) of MSN nanoparticles.

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Fig. 3. The nitrogen adsorption/desorption isotherms and pore size distributions of MSN, MSN-SH and MSN-SS-HA.

Table 1 The N2 adsorption–desorption nanoparticles.

parameters

of

different

functionalized

2.8. Cellular uptake

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A quantitative determination of the cellular uptake was performed on a fluorescence-activated cell sorter (FACS). HCT-116 and NIH-3T3 cells were seeded in 6-well plates at a density of 5  105 per well and incubated overnight. After washing with PBS, the cells were incubated with 100 and 200 lg mL 1 FITC labeled MSN-SH or MSN-SS-HA in 2 mL serum-free DMEM medium for 2 h at 37 °C or 4 °C. Subsequently, the cells in each well were rinsed with cold PBS for three times, trypsinized and resuspended in 400 lL PBS. The mean fluorescence intensity (MFI)

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MSN

Sample

SBET (m2/g)

VP (cm3/g)

WBJH (nm)

MSN MSN-SH MSN-SS-HA

1268 864 471

1.08 0.89 0.39

2.8 2.7 <2

Fig. 4. (A) The corresponding zeta potentials of MSN-SH, MSN-SS-NH2 and MSN-SS-HA; (B) the FT-IR spectra of MSN-SH (a), MSN-SS-NH2 (b) and MSN-SS-HA (c) and (C) the TGA curves of MSN-SH, MSN-SS-NH2 and MSN-SS-HA.

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Fig. 5. Schematic illustration of two different drug loading methods. (A) MSN-SH used as the initial drug carrier, and (B) MSN-SS-NH2 used as the initial drug carrier.

Fig. 6. Cumulative release profiles of MSN-SS-HA/DOX and MSN-SH/DOX in pH 5.0 PBS (A) and pH 7.4 PBS (B) under different conditions.

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was measured by FACS (FACSCalibur, Becton Dickinson, Mountainview, CA, USA). In the competition experiments, 2 mg/mL HA was applied and incubated with the HCT-116 cells for 2 h before the nanoparticles were added. For microscopic analysis, HCT-116 and NIH-3T3 cells were cultured in round glass coverslips in 24-well plates. After washing three times with PBS, FITC labeled MSN-SS-HA nanoparticles were added to the corresponding wells at a final concentration of 100 lg/mL. After an incubation period of 2 h, the cells were washed three times with cold PBS, fixed with 4% formaldehyde, and Hoechst 33258 was used to stain the nuclei. The fixed cells were observed with a Leica DM-6000 CS microscope (Leica Instruments Inc., Wetzlar, Germany).

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2.9. In vitro cytotoxicity test

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Cytotoxic activities of blank nanoparticles and DOX-loaded samples toward HCT-116 and NIH3T3 cells were evaluated by MTT assay. After the cells achieved ca. 80% confluence, the cells were trypsinized and seeded onto 96-well plates at a density of 2  104 cells/well. After a 24 h incubation, the culture medium was removed and fresh medium containing serial concentrations

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of DOX (0.01, 0.1, 1.0, 5.0, 10.0 lg/mL) formulations were added. After a 24 h incubation, 20 lL MTT solution (0.5 mg/mL) was added to the cell culture medium and incubated for 4 h to quantify the living cells. Then, the DMEM medium was removed and DMSO (150 lL) was added. Finally, the 96-well plates were placed in an iMark™ microplate reader (Bio-RAD, Hercules, CA, USA) to measure absorbance at 570 nm.

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2.10. Characterization

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The morphology and the mesoporous structure of the nanoparticles were confirmed by scanning electron microscopy (SEM) images (ZEISS, SUPRA 35, Oberkochen, Germany) and a Transmission electron microscopy (TEM) (Tecnai G2 F30, FEI, Eindhoven, The Netherlands). Nitrogen adsorption analysis was measured using an adsorption analyzer (V-Sorb 2800P, Gold APP Instrument Corporation, Beijing, China). Particle sizes and zeta potentials were measured on a Nano-zs90 Nanosizer (Malvern Instruments Ltd., Worcestershire, UK). Thermogravimetric analysis (TGA) was performed on a TGA-50 instrument (Shimadzu, Kyoto, Japan) with a heating rate of 5 °C/min under a nitrogen flow.

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Fourier transform infrared spectrophotometric (FT-IR) spectra were measured on a FT-IR spectrometer (Bruker IFS 55, Faellanden, Switzerland) using the KBr pellet technique. The investigated spectroscopic range was from 400 to 4000 cm 1.

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2.11. Statistical analysis

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Statistical analysis was carried out with Student’s t-test using SPSS software with p values <0.05 being considered as statistically significant differences.

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3. Results and discussion

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3.1. Preparation and characterization of MSN-SS-HA

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The synthetic process of the MSN-based nanoparticles with HA as caps connected via disulfide bonds is summarized in Fig. 1A. Firstly, MSN-SH was synthesized by a post-grafting method, and then reacted with Py-SS-NH2 by a disulfide bond exchange reaction to obtain MSN-SS-NH2. Finally, HA polymers were covalently grafted onto the surfaces of MSN by an amidation reaction. The average diameter of MSN nanoparticles was ca. 120 nm as shown in the SEM image and TEM image (Fig. 2). From the TEM image shown in Fig. 2B, an array of well-ordered mesopores could be clearly observed in the MSN nanoparticles. The isotherms and pore size distribution curves of MSN, MSN-SH and MSN-SS-HA were measured by N2 adsorption–desorption analysis as shown in Fig. 3. The Brunauer–Emmett–Teller (BET) surface area (SBET), total pore volume (VP), and Barrett–Joyner–Halenda (BJH) pore size distribution (WBJH) are summarized in Table 1. After the thiol modification, the WBJH was slightly reduced to 2.7 nm, which indicated that the internal mesoporous network was not blocked after the grafting of thiol on the surface of MSN. The SBET and VP were gradually reduced after different grafting processes. The successful grafting of MSN-SS-HA was validated by different methods. As shown in Fig. 4A, the zeta potential of MSN-SH was reversed from a negative value of 21.5 mV to a positive one of +34.1 mV after the formation of MSN-SS-NH2 due to the amine groups on the silica surface. After grafting HA, the zeta potential was reversed again to a negative one of 25.7 mV, confirming the successful grafting process. A comparison of the FT-IR spectra of MSN-SH, MSN-SS-NH2 and MSN-SS-HA is shown in Fig. 4B. The presence of the thiol groups on MSN-SH was confirmed by the minor peak around 2560 cm 1. After MSN-SH reacted with

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Fig. 7. Change in the zeta potential of MSN-SS-HA incubated with 150 UmL HAase.

1

Py-SS-NH2, the appearance of a minor peak around 1505 cm 1 was characteristic of the amino groups, while the minor peak of thiol groups around 2560 cm 1 disappeared, indicating the formation of MSN-SS-NH2. TGA curves of MSN-SH, MSN-SS-NH2 and MSN-SS-HA are shown in Fig. 4C. Regarding MSN-SS-HA, an additional weight loss of about 11% could be ascribed to the removal of the HA polymer on the surface of silica.

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3.2. Drug loading efficiency of different drug loading methods

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In order to achieve a relatively high drug loading efficiency, two different drug loading methods were used to load DOX as described in Fig. 5. In the first loading method, MSN-SH was used as the initial drug carrier. The zeta potential of MSN-SH was 21.5 mV, and DOX had a positive charge in PBS solution with a pKa of 8.2. Therefore, MSN-SH had a strong interaction with DOX in pH 7.4 PBS due to electrostatic interactions, and the drug loading efficiency of MSN-SH/DOX was 19.8%. After MSN-SH/DOX reacted with Py-SS-NH2, the amine groups on the surface of MSN would delay the release of DOX during grafting of HA. Also, MSN-SS-HA/DOX had a relatively high drug loading efficiency up to 10.6%. In contrast, in the second drug loading method, when MSN-SS-NH2 was used as the initial carrier, the positively charged DOX was difficult to load into the mesopores of silica due to the electrostatic repulsion. So, the drug loading efficiency was less than 3%. Furthermore, the SBET of MSN-SS-HA/DOX was markedly reduced compared with that of MSN-SS-HA/DOX-2. It is worth mentioning that HA modified MSNs (HA-MSNs) for targeted drug delivery have been reported by Yu and co-workers [23]. DOX was used as a model drug, and the drug loading efficiency of MSNs was up to 26.8%. However, the loading efficiency of HA-MSNs was only 1.2% by mixing PBS solution containing HA-MSNs with DOX-PBS solution (1 mg/mL). Hence, the optimal drug loading method used MSN-SH as the initial drug carrier. The existing state of DOX in MSN-SS-HA/DOX and MSN-SS-HA/DOX-2 was both in a non-crystalline state due to the confined effect of the mesopore of the silica as shown in Fig. S1 [24].

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3.3. Stimuli-responsive drug release

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To investigate the redox and enzyme dual-stimuli responsive release of the MSN-SS-HA, DOX-loaded MSN-SS-HA/DOX samples were added to pH 5.0 and pH 7.4 PBS at different conditions of GSH and HAase. As shown in Fig. 6A, MSN-SH/DOX exhibited a relatively fast release of DOX in pH 5.0 PBS, and the cumulative release was more than 70% within a period of 48 h. However, less than 20% DOX was released from MSN-SS-HA/DOX into fluids in the absence of GSH and HAase in pH 5.0 PBS for a period of 48 h, indicating the good capping efficiency obtained using HA chains and disulfide bonds. Nevertheless, in the presence of HAase or GSH, the cumulative release was markedly increased to ca. 30% and 50% within a period of 48 h, respectively. In addition, the cumulative release was further improved to 60% in the simultaneous presence of GSH and HAase. The relatively slow release and low cumulative release percent during the 48 h were due to the electrostatic interaction between the positive charged DOX and the negative charged silica[25] as well as the drug-wall attractions (drug release kinetics on pore size) which have been discussed in detail in the recent literatures [26,27]. The same tendency was also seen in pH 7.4 PBS (Fig. 6B). The release rate was relatively low (less than 8% in 24 h) in pH 7.4 PBS due to the stronger electrostatic interaction between DOX and silica compared with that in pH 5.0 PBS. However, the cumulative release was markedly increased in the presence of HAase or GSH. Furthermore, the cumulative release percentage of DOX was further improved to ca. 30% in the simultaneous presence of GSH and HAase.

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Fig. 8. (A) Size distribution of MSN-SH and MSN-SS-HA; (B) images of MSN-SH and MSN-SS-HA dispersed in pH 7.4 PBS with a SiO2 concentration of 5 mg/mL.

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The reason for the increasing release rate of MSN-SS-HA/DOX under GSH/HAase is as follows. The disulfide bonds of MSN-SS-HA were cleaved under high concentrations of GSH, which allowed the separation of the polymer from MSN-SS-HA, so DOX was rapidly released from MSN-SS-HA/DOX. In the presence of HAase, the outer layer macromolecular HA was degraded by HAase into small fragments, which allowed some of the DOX to be released from the carrier. However, because of the residual small fragments of HA and disulfide bonds, the release rate of DOX was slower than that in the presence of GSH. In addition, in the simultaneous presence of GSH and HAase, when the surface macromolecular HA of MSN-SS-HA was degraded by HAase into small fragments, the disulfide bonds of MSN-SS-HA were more easily cleaved than in the presence of GSH alone. Hence, MSN-SS-HA/DOX exhibited the fastest release rate in the simultaneous presence of GSH and HAase in pH 5.0 PBS and pH 7.4 PBS.

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3.4. Degradation of HA by HAase

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As shown above in Fig. 4A, MSN-SS-HA has a negative charge at 25.7 mV, while the MSN-SS-NH2 sample without HA grafting has a highly positive charge +34.1 mV. The amino groups exposed on the outer surface of silica contributed to the positive zeta potential. After grafting with HA, the amino groups partially reacted with the carboxylic acid of HA to form amides, and the small quantity of residual amines was covered by the surface chains of HA. To confirm the degradation of HA on the surface of MSN-SS-HA, the zeta potential of the sample was measured in the presence of HAase in pH 5.0 PBS and pH 7.4 PBS. As shown in Fig. 7, the zeta potential of MSN-SS-HA increased with the prolonged incubation time in the presence of HAase regardless of the pH values. Moreover, the change of zeta potential in pH 5.0 PBS was greater than that in pH 7.4 PBS, which is attributed to the fact that the hydrolysis ability of HAase was enhanced under increasingly acidic conditions.

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These results demonstrated that the modified long-chain HA on the surface of MSN is hydrolyzed by HAase.

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3.5. The advantage of MSN-SS-HA after modification of HA

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Although MSN are versatile and promising materials for targeted and stimuli-responsive drug delivery, the aggregation phenomenon under physiological conditions, the nonspecific binding in the blood and hemolytic characteristics limit their applications in nanomedicine [28]. To address this challenge, the strategy of HA-conjugated MSN was developed to increase the physiological stability and biocompatibility. The hydrodynamic diameter of MSN-SH and MSN-SS-HA was measured in distilled water (Fig. 8A). The diameter of MSN-SH was 187 nm with a polydispersity index (PDI) of 0.134. Although MSN-SS-HA had a greater diameter of 256 nm than that of MSN-SH due to the grafting of HA chains, the PDI of MSN-SS-HA (0.049) was much lower than that of MSN-SH. Compared with TEM data, the diameters of MSN-SH and MSN-SS-HA measured by dynamic light scattering (DLS) were slightly larger than those of TEM results. TEM provided the size of the nanoparticles in a dried state without the hydration layer. However, the hydrodynamic diameter of the nanoparticles was measured in water conditions. The hydrodynamic diameter of MSN-SS-HA measured by DLS corresponds to the diameter of the dense silica core, plus the thickness of the hydration layer arising from the hydrophilic HA on the surface of MSN. As shown in Fig. 8B, MSN-SH showed poor stability in physiological saline and precipitated rapidly after dispersion. In contrast, the dispersibility of MSN-SS-HA was dramatically improved after conjugation with HA. MSN-SS-HA nanoparticles can remain stable in physiological PBS for more than 24 h. The excellent dispersibility of MSN-SS-HA could be attributed to the electrostatic repulsions and steric hindrance among the stretched hydrophilic HA chains. In addition, the hemolytic behavior and BSA adsorption were also examined to evaluate the biocompatibility of MSN-SH and

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Fig. 9. (A) Confocal microscopy images of CD44 receptor-mediated endocytosis of FITC labeled MSN-SS-HA by HCT-116 and NIH-3T3 cells. (B) The MFI of FITC labeled MSNSH and MSN-SS-HA was measured by FACS in HCT-116 and NIH-3T3 cells. Data are expressed as mean ± SD (n = 3). ⁄⁄p < 0.01.

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MSN-SS-HA. The percentage hemolysis was markedly decreased from 45.9% for MSN-SH to less than 1% after the modification of HA at the extremely high concentration of 1500 lg
mL 1 as shown in Fig. S2. Also, the adsorption of BSA was sharply reduced from 1.93 wt.% to 0.27 wt.% after the modification of HA. Therefore, the MSN-SS-HA nanoconjugates exhibited long-term stability under physiological conditions and a reduced percentage hemolysis as well as BSA adsorption, which were beneficial for improving active target ability and an enhanced permeation and retention (EPR) effect by prolonging the blood circulation time.

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3.6. In vitro targeting analysis of MSN-SS-HA nanoparticles

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CD44 receptors over-expressed HCT-116 cells and receptor-negative NIH-3T3 cell lines were used to evaluate the

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cellular uptake of MSN-SS-HA nanoparticles. As shown in Fig. 9A, the fluorescence originating from FITC-labeled MSN-SS-HA was higher in HCT-116 cells than that in NIH-3T3 cells, indicating that MSN-SS-HA was readily taken up by HCT-116 cells. The cellular uptake performance was further studied by FACS analysis to obtain a quantitative comparison between FITC labeled MSN-SS-HA and MSN-SH nanoparticles. Fig. 9B showed that the cellular uptake of MSN-SH and MSN-SS-HA in HCT-116 and NIH-3T3 cells occurred in a concentration-dependent manner. There was no obvious difference in MFI of MSN-SH between HCT-116 and NIH-3T3 cells at concentrations of 100 and 200 lg mL 1. Nevertheless, the MFI of MSN-SS-HA increased 3.0-fold and 2.7-fold for 100 and 200 lg mL 1 in HCT-116 cells compared with that of MFI in NIH-3T3 cells. Surprisingly, the MFI of MSN-SS-HA was markedly weaker compared with that of MSN-SH in NIH-3T3 cells, which

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Fig. 10. Intracellular MFI of blank cells (Blank), cells with FITC-labeled MSN-SS-HA (MSN-SS-HA), cell preincubated with HA before the addition of FITC-labeled MSNSS-HA (MSN-SS-HA+HA) and cells with FITC-labeled MSN-SS-HA kept at 4 °C (MSNSS-HA at 4 °C) in HCT-116 cells were analyzed by FACS.

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may be due to the fact that the fluorescence intensity of FITC labeled MSN-SS-HA was reduced compared with FITC labeled MSN-SH after the grafting of Py-SS-NH2 and HA. The reduced fluorescence was due to the increased weight after the grafting and the fraction of quenched fluorescence after the two-step modification process[29]. These results indicated that the modified HA on the surface of MSN nanoparticles might improve the cellular uptake of MSN-SS-HA in CD44 receptors over-expressed HCT-116 cell. The addition of free HA and low temperature were used to further confirm that the target-specific endocytosis of FITC-labeled MSN-SS-HA was mediated by CD44 receptors. As shown in Fig. 10, the MFI was reduced to half that of the untreated group, when HCT-116 cells were pretreated with 2 mg/mL HA for 2 h. In addition, the experiment performed at 4 °C showed that the cellular uptake of MSN-SS-HA was markedly inhibited, because receptor-mediated uptake involves ATP-dependent endocytosis and is relatively inactive at low temperatures. Therefore, the apparent suppression of cellular uptake by free HA or low temperatures demonstrated that the endocytosis of MSN-SS-HA was mainly mediated by CD44 receptors. In addition, MSN-SS-HA/DOX was also investigated by using CLSM to further demonstrate the enhanced uptake of DOX in HCT-116 cells as shown in Fig. S3.

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3.7. In vitro cytotoxicity studies of MSN-SS-HA/DOX and MSN-SH/DOX

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The cell viability of HCT-116 and NIH-3T3 cells incubated with blank MSN-SS-HA and MSN-SH nanoparticles for 48 h was above 80% within the tested concentration range as shown in Fig. S4, demonstrating the good safety of the carriers. The biological activity of released DOX from MSN-SS-HA/DOX was evaluated by the MTT assay. The cell viability of HCT-116 and NIH-3T3 cells incubated with raw DOX and released DOX for 48 h were not significantly different as shown in Fig. S5. These results indicated that the biological activity of released DOX did not alter by the drug loading and release. HCT-116 and NIH-3T3 cells incubated with free DOX, MSN-SS-HA/DOX and MSN-SH/DOX showed a DOX dose-dependent cytotoxicity at a series of DOX concentrations from 0.01 to 10 mg/mL (Fig. 11A and B). In CD44 receptor-positive HCT-116 cells, the formulation of MSN-SS-HA/DOX exhibited a higher cytotoxicity compared with that of MSN-SH/DOX, indicating that the promotion of internalization of the MSN-SS-HA nanoparticles via CD44 receptor-mediated endocytosis increased the antitumor effect of DOX. Instead, in the receptor-negative group (NIH-3T3 cells), the poor cellular uptake and incomplete release of drugs from MSN-SS-HA reduced the efficacy of DOX. Furthermore, the anticancer effect of DOX was quantified by the IC50 (the concentration at which 50% of cells were killed). As shown in Table 2, the anticancer effects of DOX were increased slightly by MSN-SS-HA/DOX nanoparticles, compared with free DOX and MSN-SH/DOX in HCT-116 cancer cells. However, for NIH-3T3 cells, the IC50 value increased from 0.8 lg/mL for free DOX to 4.5 lg/mL for the MSN-SS-HA/DOX formulation. In addition, the IC50 value of MSN-SS-HA/DOX was reduced from 4.5 lg/mL in NIH-3T3 cells to 0.6 lg/mL in HCT-116 cells. Therefore, MSN-SS-HA/DOX nanocarriers could increase the anticancer effects of DOX in CD44 receptor-positive tumor cells and reduce the side effects in CD44 receptor-negative cells.

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4. Conclusions

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In summary, a redox/enzyme dual-stimuli responsive targeted delivery system has been developed, in which HA was grafted on the surface of MSN by disulfide bonds. MSN-SH was used as the initial carrier to load DOX. The MSN-SS-HA/DOX had a relatively high drug loading efficiency up to 12.5%. In vitro release profiles showed that the release of DOX was markedly restricted in the absence of GSH and HAase, while it was markedly accelerated upon the addition of GSH/HAase. Furthermore, the release was further accelerated in the simultaneous presence of GSH and HAase. In

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Fig. 11. Cytotoxicity of free DOX, MSN-SS-HA/DOX and MSN-SH/DOX against HCT-116 (A) and NIH-3T3 (B) cells at different concentrations.

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Table 2 IC50 value for different DOX loaded samples and free DOX for inhibiting growth of HCT-116 and NIH-3T3 cells after 48 h incubation. Formulations

Free DOX MSN-SS-HA/DOX MSN-SH/DOX

IC50 (lg/mL) HCT-116

NIH-3T3

0.8 0.6 1.9

0.8 4.5 1.9

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vitro targeting studies showed that MSN-SS-HA exhibited higher uptake efficiency in CD44 receptor over-expressed HCT-116 cells via CD44 receptor-mediated endocytosis. In vitro cytotoxicity studies showed that MSN-SS-HA/DOX was more cytotoxic toward HCT-116 cells than NIH-3T3 due to the increased cellular uptake. The results of this study suggest a potential strategy for dual-stimuli responsive targeted cancer therapy.

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5. Disclosures

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We have no conflicts of interest.

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Acknowledgements

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This work was supported by National Basic Research Program of China (973 Program) (No. 2015CB932100) and National Natural Science Foundation of China (No. 81473165).

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Appendix A. Figures with essential color discrimination

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Certain figures in this article, particularly Figs. 1–9 and 11, 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.2015.05.010.

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Appendix B. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.05. 010.

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References

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[1] T.-W. Kim, I.I. Slowing, P.-W. Chung, V.S.-Y. Lin, Ordered mesoporous polymersilica hybrid nanoparticles as vehicles for the intracellular controlled release of macromolecules, ACS Nano 5 (2010) 360–366. [2] C.H. Lee, S.H. Cheng, I. Huang, J.S. Souris, C.S. Yang, C.Y. Mou, et al., Intracellular pH-responsive mesoporous silica nanoparticles for the controlled release of anticancer chemotherapeutics, Angew Chem 122 (2010) 8390–8395. [3] W. Chen, P. Zhong, F. Meng, R. Cheng, C. Deng, J. Feijen, et al., Redox and pHresponsive degradable micelles for dually activated intracellular anticancer drug release, J Control Release 167 (2013) 171–179. [4] X. Wang, X. Cai, J. Hu, N. Shao, F. Wang, Q. Zhang, et al., Glutathione-triggered ‘‘Off–On’’ release of anticancer drugs from dendrimer-encapsulated gold nanoparticles, J Am Chem Soc 135 (2013) 9805–9810. [5] R. Guo, L.L. Li, W.H. Zhao, Y.X. Chen, X.Z. Wang, C.J. Fang, et al., The intracellular controlled release from bioresponsive mesoporous silica with folate as both targeting and capping agent, Nanoscale 4 (2012) 3577–3583.

[6] C. Park, H. Kim, S. Kim, C. Kim, Enzyme responsive nanocontainers with cyclodextrin gatekeepers and synergistic effects in release of guests, J Am Chem Soc 131 (2009) 16614–16615. [7] D. He, X. He, K. Wang, J. Cao, Y. Zhao, A light-responsive reversible moleculegated system using thymine-modified mesoporous silica nanoparticles, Langmuir 28 (2012) 4003–4008. [8] R. Cheng, F. Feng, F. Meng, C. Deng, J. Feijen, Z. Zhong, Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery, J Control Release 152 (2011) 2–12. [9] Y. Cui, H. Dong, X. Cai, D. Wang, Y. Li, Mesoporous silica nanoparticles capped with disulfide-linked PEG gatekeepers for glutathione-mediated controlled release, ACS Appl Mater Interfaces 4 (2012) 3177–3183. [10] G. Saito, J.A. Swanson, K.-D. Lee, Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities, Adv Drug Deliv Rev 55 (2003) 199–215. [11] R. Cheng, F. Meng, C. Deng, H.-A. Klok, Z. Zhong, Dual and multi-stimuli responsive polymeric nanoparticles for programmed site-specific drug delivery, Biomaterials 34 (2013) 3647–3657. [12] C. Chen, J. Geng, F. Pu, X. Yang, J. Ren, X. Qu, Polyvalent nucleic acid/ mesoporous silica nanoparticle conjugates: dual stimuli-responsive vehicles for intracellular drug delivery, Angew Chem Int Ed 50 (2011) 882–886. [13] Y. Zhao, J.L. Vivero-Escoto, I.I. Slowing, B.G. Trewyn, V.S. Lin, Capped mesoporous silica nanoparticles as stimuli-responsive controlled release systems for intracellular drug/gene delivery, Expert Opin Drug Deliv 7 (2010) 1013–1029. [14] R. Stern, M.J. Jedrzejas, Hyaluronidases: their genomics, structures, and mechanisms of action, Chem Rev 106 (2006) 818–839. [15] B.P. Toole, Hyaluronan: from extracellular glue to pericellular cue, Nat Rev Cancer 4 (2004) 528–539. [16] K.Y. Choi, H.Y. Yoon, J.-H. Kim, S.M. Bae, R.-W. Park, Y.M. Kang, et al., Smart nanocarrier based on PEGylated hyaluronic acid for cancer therapy, ACS Nano 5 (2011) 8591–8599. [17] K.L. Chao, L. Muthukumar, O. Herzberg, Structure of human hyaluronidase-1, a hyaluronan hydrolyzing enzyme involved in tumor growth and angiogenesis, Biochemistry 46 (2007) 6911–6920. [18] C. Surace, S. Arpicco, Dufaÿ.-Wojcicki. Al, Marsaud. Vr, Bouclier. Cl, D. Clay, et al., Lipoplexes targeting the CD44 hyaluronic acid receptor for efficient transfection of breast cancer cells, Mol Pharm 6 (2009) 1062–1073. [19] I. Rivkin, K. Cohen, J. Koffler, D. Melikhov, D. Peer, R. Margalit, Paclitaxelclusters coated with hyaluronan as selective tumor-targeted nanovectors, Biomaterials 31 (2010) 7106–7114. [20] H.-J. Cho, H.Y. Yoon, H. Koo, S.-H. Ko, J.-S. Shim, J.-H. Lee, et al., Self-assembled nanoparticles based on hyaluronic acid-ceramide (HA-CE) and PluronicÒ for tumor-targeted delivery of docetaxel, Biomaterials 32 (2011) 7181–7190. [21] Z. Chen, Z. Li, Y. Lin, M. Yin, J. Ren, X. Qu, Bioresponsive hyaluronic acid-capped mesoporous silica nanoparticles for targeted drug delivery, Chem-Eur J 19 (2013) 1778–1783. [22] Y.W. Ebright, Y. Chen, Y. Kim, R.H. Ebright, S-[2-(4-A zidosalicylamido) ethylthio]-2-thiopyridine: radioiodinatable, cleavable, photoactivatible crosslinking agent, Bioconjugate Chem 7 (1996) 380–384. [23] M. Yu, S. Jambhrunkar, P. Thorn, J. Chen, W. Gu, C. Yu, Hyaluronic acid modified mesoporous silica nanoparticles for targeted drug delivery to CD44overexpressing cancer cells, Nanoscale 5 (2013) 178–183. [24] T. Ukmar, A. Godec, O. Planinšek, V. Kaucˇicˇ, G. Mali, M. Gaberšcˇek, The phase (trans) formation and physical state of a model drug in mesoscopic confinement, Phys Chem Chem Phys 13 (2011) 16046–16054. [25] H. Tang, J. Guo, Y. Sun, B. Chang, Q. Ren, W. Yang, Facile synthesis of pH sensitive polymer-coated mesoporous silica nanoparticles and their application in drug delivery, Int J Pharm 421 (2011) 388–396. [26] T. Ukmar, U. Maver, O. Planinšek, V. Kaucˇicˇ, M. Gaberšcˇek, A. Godec, Understanding controlled drug release from mesoporous silicates: theory and experiment, J Control Release 155 (2011) 409–417. [27] T. Ukmar, M. Gaberšcˇek, F. Merzel, A. Godec, Modus operandi of controlled release from mesoporous matrices: a theoretical perspective, Phys Chem Chem Phys 13 (2011) 15311–15317. [28] L.-S. Wang, L.-C. Wu, S.-Y. Lu, L.-L. Chang, I.-T. Teng, C.-M. Yang, et al., Biofunctionalized phospholipid-capped mesoporous silica nanoshuttles for targeted drug delivery: improved water suspensibility and decreased nonspecific protein binding, ACS Nano 4 (2010) 4371–4379. [29] Q. Zhao, H. Geng, Y. Wang, Y. Gao, J. Huang, Y. Wang, et al., Hyaluronic acid oligosaccharide modified redox-responsive mesoporous silica nanoparticles for targeted drug delivery, ACS Appl Mater Interfaces 6 (2014) 20290–20299.

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