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IJP 14880 1–13 International Journal of Pharmaceutics xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Multifunctional mesoporous silica nanoparticles mediated co-delivery of paclitaxel and tetrandrine for overcoming multidrug resistance Lejiao Jia a , Zhenyu Li b , Jingyi Shen c , Dandan Zheng c , Xiaona Tian c, Hejian Guo c , Ping Chang a, * a b c

Department of Pharmacy, Qilu Hospital of Shandong University, 107 Wenhua Xilu, Jinan 250012, PR China Key Laboratory of Chemical Biology of Ministry of Education, College of Pharmacy, Shandong University, 44 Wenhua Xilu, Jinan 250012, PR China Department of Pharmaceutics, College of Pharmacy, Shandong University, 44 Wenhua Xilu, Jinan 250012, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 January 2015 Received in revised form 15 April 2015 Accepted 4 May 2015 Available online xxx

The objective of the study is to fabricate multifunctional mesoporous silica nanoparticles for achieving co-delivery of conventional antitumor drug paclitaxel (PTX) and the multidrug resistance reversal agent tetrandrine (TET) expecting to overcome multidrug resistance of MCF-7/ADR cells. The nanoparticles were facile to prepare by self-assemble in situ drug loading approach. Namely, PTX and TET were solubilized in the cetyltrimethylammonium bromide (CTAB) micelles and simultaneously silica resources hydrolyze and condense to form nanoparticles. The obtained nanoparticles, denoted as PTX/TETCTAB@MSN, exhibited pH-responsive release property with more easily released in the weak acidic environment. Studies on cellular uptake of nanoparticles demonstrated TET could markedly increase intracellular accumulation of nanoparticles. Furthermore, the PTX/TET-CTAB@MSN suppressed tumor cells growth more efficiently than only delivery of PTX (PTX-CTAB@MSN) or the free PTX. Moreover, the nanoparticle loading drugs with a PTX/TET molar ratio of 4.4:1 completely reversed the resistance of MCF-7/ADR cells to PTX and the resistance reversion index was 72.3. Mechanism research showed that both TET and CTAB could arrest MCF-7/ADR cells at G1 phase; and besides PTX arrested cells at G2 phase. This nanocarrier might have important potential in clinical implications for co-delivery of multiple drugs to overcome MDR. ã 2015 Published by Elsevier B.V.

Keywords: Mesoporous silica nanoparticles Paclitaxel Tetrandrine Co-delivery Multidrug resistance

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1. Introduction

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Multidrug resistance (MDR) is one of the main obstacles compromising the efficacy of chemotherapy treatment of tumors. The term MDR is defined as a resistance phenotype where tumor cells become resistant simultaneously to multiple drugs with no obvious structural resemblance and with different molecular targets (Eytan, 2005; Larsen et al., 2000). Although the etiology of MDR is multifactorial, the overexpression of P-glycoprotein (P-gp) is believed as the major cause. P-gp, coded by the mdr1 gene, belongs to the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) transporters and functions as an energy-dependent drug efflux pump that can actively pump out a variety of hydrophobic anticancer drugs from the target cancer cells and hence reduces the intracellular accumulation of drugs (Gottesman et al., 2002; Loo and Clarke, 2005). The recognition of

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* Corresponding author. Tel.: +86 531 82169668; fax: +86 531 82169668. E-mail address: [email protected] (D. Zheng).

P-gp-mediated MDR has provided the impetus for discovering effective strategies to overcome MDR. To date, these strategies are mainly involved in silencing the expression of the efflux transporter through RNA interference or using functional inhibitors, also called chemosensitizers (Fan et al., 2010; Yu et al., 2012; Q2 Zhu et al., 2010). P-gp-mediated drug efflux could be inhibited or altered by a wide range of chemosensitizers. Verapamil, a calcium channel blocker, was the first discovered P-gp inhibitor. Since then, various classes of compounds were brought to light such as first-generation P-gp inhibitors (cyclosporine A and quinidine), second-generation reversal agents (elacridar, valspodar and dexverapamil) and third-generation P-gp inhibitors (zosuquidar, tariquidar and laniquidar) (Eckford and Sharom, 2009). However, lots of these compounds are ineffective or show serious toxicity at the doses required to inhibit P-gp function, or induced unfavorable pharmacokinetic interactions, resulting in failure in their clinical trials (Fisher et al., 1996; Fracasso et al., 2001; Georges et al., 1990; Pennock et al., 1991). Therefore, novel and more potent reversal agents of MDR are still needed. Tetrandrine (TET), a

http://dx.doi.org/10.1016/j.ijpharm.2015.05.010 0378-5173/ ã 2015 Published by Elsevier B.V.

Please cite this article in press as: Jia, L., et al., Multifunctional mesoporous silica nanoparticles mediated co-delivery of paclitaxel and tetrandrine for overcoming multidrug resistance. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.05.010

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bis-benzylisoquinoline alkaloid, is isolated from the roots of Chinese traditional herb “Hanfangji” (Radix Stephania tetrandrae S. Moore). TET has been used to treatment of hypertension, inflammation, silicosis and angina pectoris in China since the 1950s (Qian, 2002; Xie et al., 2002). It is relatively non-toxic to humans, even at the administration of 180 mg, intramuscularly (i. m.) three times daily (t.i.d.) (Chen, 2002; Dai et al., 2007). Recent studies have shown that TET has a markedly reversal effect on Pgp-mediated MDR in vitro and in vivo (Fu et al., 2004; Zhu et al., 2005). The purpose of the present study is to co-administration of conventional antitumor drug paclitaxel (PTX) with the MDR reversal agent TET expecting to increase the intracellular paclitaxel concentration and enhance its antitumor effect. However, both of the two drugs are water-insoluble. Besides, it is necessary to minimize the exposure of normal cells to inhibitor and anticancer drug, and to co-localize both them in tumor cells. Nanotechnology has shown tremendous process in drug delivery. Currently, the silica-based nanomaterials, such as sol–gel, colloidal, and mesoporous silica nanoparticles (MSNs), have become very active in controlled release, drug delivery, MDR reversing and other biotechnological applications (Ashley et al., 2012; Chen et al., 2013, 2014a,b, 2012; Liu et al., 2012; Thomas et al., 2010; Trewyn et al., 2007). MSNs possess some attractive features for application in the delivery of drugs such as easily tuning particle/pore size, the high surface area and pore volume, as well as being biocompatible and chemically inert (Tang et al., 2012; Zhang et al., 2012). The MSNs before removing the surfactant templates are regarded as a novel kind of organic–inorganic composite nano-delivery system (He et al., 2010). In such a drug delivery system, drugs can be solubilized in the surfactant micelles and simultaneously silica resources hydrolyze and condense to form nanoparticles. In this work, cetyltrimethylammonium bromide (CTAB) was chosen as the surfactant template. CTAB is a known component of the broadspectrum antiseptic cetrimide, which has demonstrated anticancer properties in vitro and in vivo by targeting tumor mitochondria (Bleday et al., 1986; Weiss et al., 1987; Yip et al., 2006). Herein, our obtained multifunctional mesoporous silica nanoparticles, defined as PTX/TET-CTAB@MSN, possess the following advantages. The drugs PTX and TET are solubilized in CTAB micelles of nanoparticles and thus they can be co-delivered to the tumor cells. TET functions as chemosensitizer, which could inhibit the drug efflux pump (P-gp) to increase the intracellular accumulation of PTX and CTAB, resulting in enhancement of their antitumor activities. Moreover, the PTX/TET-CTAB@MSN exhibits pH-responsive drug release behavior due to that the CTAB micelles could be destroyed and then loaded drugs leak in low pH environment, while drugs are hardly released in neutral environment. It is well known that pH in sites of inflammation, diseased tissues and tumors is more acidic (pH 6.5–7.4) than that in normal tissue (pH 7.4). Besides, intracellular pH varies depending on organelle. Endosomes and lysosomes typically exhibit pH values of 6.8–4.5 (Liechty and Peppas, 2012; Zhao et al., 2012). Therefore, PTX/TET-CTAB@MSN can deliver drugs targeting to tumors and reduce the toxicity in normal tissues. 2. Materials and methods 2.1. Materials Cetyltrimethylammonium bromide (CTAB, 99%) and tetraethyl orthosilicate (TEOS, 98%) were purchased from Alfa Aesar (Heysham, Lancs). Paclitaxel (PTX) was obtained from Xi’an Haoxuan Bio-tech Co., Ltd. (Xi’an, China). Tetrandrine and 3-aminopropyltriethoxysilane (APTES) were purchased from Aladdin (Shanghai, China). Ammonium fluoride (NH4F, 96%) was

provided by Sinopharm chemical reagent Co., Ltd. (Shanghai, China). Fluorescein isothiocyanate (FITC) was purchased from Klontech (Jinan, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT), dimethyl sulfoxide (DMSO), Hoechst 33342 and propidium iodide (PI) were obtained from Sigma Co., Ltd. (USA). Penicilline-streptomycin, RPMI1640, fetal bovine serum (FBS) and 0.25% (w/v) trypsin solution were purchased from Gibco BRL (Gaithersberg, MD, USA). Annexin V-FITC Apoptosis Detection kit was obtained from KeyGen Bio-tech Co., Ltd. (Nanjing, China). All other chemicals were of reagent grade and the deionized water generated using Millipore Milli-Q system (Billerica, MA).

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2.2. Cell line

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Human breast cancer MCF-7 cell and its multidrug-resistant cell (MCF-7/ADR) were kindly donated by the Department of Pharmacology, School of Medicine, Shandong University. The cells were cultured in RPMI1640 medium, supplemented with 10% FBS, 100 U/mL of penicillin and 100 mg/mL of streptomycin. All the cells were cultured in incubators maintained at 37
C with 5% CO2 under fully humidified conditions. All experiments were performed on cells in the logarithmic phase of growth.

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2.3. Preparation of drug-loaded nanoparticles

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The nanoparticles (PTX/TET-CTAB@MSN) were prepared by self-assemble in situ drug loading method, and TEOS and NH4F were chosen as silica resource and basic catalyst, respectively. The details are as follows. The anticancer drug paclitaxel and chemosensitizer tetrandrine were added into the mixture of distilled water (50 mL) and CTAB (100 mg); the mixture was heated to 75
C and stirred vigorously. When a clear solution was obtained, NH4F was added to accelerate the hydrolyzation and condensation of silica resources. After adding 0.36 mL of TEOS, the whole reaction was stirred for 30 min under a lucifugal condition. Subsequently, nanoparticles were collected by centrifugation (14,000 rpm, 10 min). To remove the unloaded drugs and CTAB, the obtained nanoparticles were washed by water and ethanol through centrifugation. Finally, the drugs-loaded nanoparticles were re-dispersed in water and lyophilized for further study. The preparation process of PTX-CTAB@MSN, TET-CTAB@MSN and CTAB@MSN was the same as described above, except that only PTX, TET or no drugs was added into the surfactant mixture, respectively. The control sample blank MSN was obtained as follows. 100 mg of CTAB@MSN was dispersed in a mixture of ethanol (40 mL) and 11.9 M hydrochloric acid (5 mL) and the mixture was refluxed to remove the surfactants from the pores of CTAB@MSN. After 24 h, the suspensions were centrifuged; the obtained MSN were washed thoroughly to remove the residual CTAB and then lyophilized for further study.

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2.4. Drug loading determination

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To determine the drug loading efficiency of nanoparticles, 10 mg of loaded samples were added in 5 mL methanol and ultrasonicated to let drug release from nanoparticles and then centrifuged at 14,000 rpm for 10 min. This process was repeated three times in order to let drugs thoroughly release from pores. The supernatant was collected and determined by HPLC methods. The HPLC system was Agilent 1100 series (Agilent, USA) and the chromatography column was Phenomenex-ODS column (150 mm  4.60 mm, 5 mm). The mobile phase for PTX consisted of acetonitrile (HPLC grade, TJSHIELD, China)/double-distilled water (45/55, v/v) pumped at a flow rate of 1.0 mL/min with determination wavelength of 227 nm. The mobile phase for detecting TET consisted of methanol (HPLC

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grade, TJSHIELD, China)/double-distilled water/triethylamine (80:20:0.03, v/v/v) pumped at a flow rate of 1.0 mL/min with determination wavelength of 282 nm. The following equation was applied to calculate the drug loading content: Drug loading contentð%Þ ¼

weight of the drug in a nanoparticles  100% weight of the drug  loaded nanoparticles

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2.5. The morphology and particle size analysis

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The morphology of nanoparticles was characterized by transmission electron microscope (TEM). A drop of nanoparticles was spread on a 200-mesh copper grid and dried at room temperature, and then observed by TEM (JEM-1011, JEOL, Japan). The particle sizes of nanoparticles were measured by the nanoparticle sizing measurement (Delsa nano, Beckman Coulter, USA). All samples were diluted with double distilled water to have a suitable concentration to test and each sample was determined in triplicate.

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2.6. X-ray diffraction (XRD) studies

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XRD patterns of the samples were collected using an X-ray diffractometer (D/max g-B, Rigaku, Japan). A Cu Ka radiation at 40 kV and 100 mA was used. Diffractograms were performed from the initial angle 2u = 3
to the final angle 2u = 45
with the steps of 0.02
, at a scanning speed of 4
/min (2u).

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2.7. In vitro drug release assay

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One of the requirements to conduct an appropriate drug release study is to use a sufficient volume of release medium, which should be able to dissolve the expected amount of drug released form formulations (D'Souza and DeLuca, 2006). Due to the poor aqueous solubility of PTX and TET, Tween 80 is added into release medium to create the “sink condition” and the release mediums are phosphate buffer solutions (PBS, pH 7.4, 7.0, 6.5) and acetic buffer solutions (ABS, pH 5.0) containing 0.5% (w/v) Tween 80 to simulate normal and tumor environments. The drugs release from nanoparticles was (were) investigated by the dialysis method. Briefly, 2 mL of drug-loaded nanoparticles suspension (containing 0.4 mg drugs) was placed into a preswelled dialysis bag with an 8000–12000 Da molecular weight cutoff and then the sealed dialysis bags were immersed into 20 mL of release medium in 50-mL tubes. These tubes were shaken at 37
C with a speed of 100 rpm under a light-sealed condition. At predetermined sampling time, 1 mL of samples was withdrawn from the incubation medium and measured by HPLC methods as described above. After sampling, an equal volume of fresh PBS or ABS was immediately added into the release medium. The drug release studies were carried out for 72 h.

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2.8. In vitro intracellular uptake PTX/TET-CTAB@MSN in MCF-7 and MCF-7/ADR cells Cellular uptake and distribution of PTX/TET-CTAB@MSN were studied via confocal microscopy and fluorescein isothiocyanate (FITC) was used as fluorescence probe to labeled the nanoparticles. In brief, 1 mg of FITC was mixed with 2.4 mL of APTES in 0.5 mL of absolute ethanol and stirred for 2 h under a nitrogen atmosphere and light-sealed condition. The next process was the same as described in Section 2.4 except the silica resource. Namely, TEOS was first added into the mixture, and then FITC-APTES stock

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solution was added after 15 min. The mixture was stirred for another 15 min and the FITC-labeled nanoparticles were then collected by centrifugation (14,000 rpm, 10 min) and washed with ethanol several times to remove the unreacted FITC-APTES. MCF-7/ADR cells were seeded onto an six-well chamber slices at a density of 3  105 cells per well and incubated in RPM1640 medium with 10% FBS for 24 h. Then the medium was carefully aspirated and replaced by 1 mL of fresh medium containing 25 mg/mL FITC-labeled nanoparticles. After incubation for 4 h, the medium was aspirated and cells were washed three times by phosphate buffer saline to remove the residual nanoparticles. Then, the cells were fixed by 4% paraformaldehyde at room temperature for 15 min. After washing with PBS, the nucleus was stained by Hoechst 33342. Subsequently, sample slices were visualized under a confocal microscope (LSM780, Carl Zeiss AG). High-magnification images were obtained with a 63 objective. Optical sections were averaged 2–4 times to reduce noise.

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2.9. In vitro anti-tumor studies

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To assess the cytotoxicity of TET, blank MSNs and CTAB@MSN, both MCF-7 and MCF-7/ADR cells were treated for 72 h by free TET with the concentration of 0.3–10 mM, blank MSNs with the concentration of 5–80 mg/mL and CTAB@MSN with the concentration of 5–80 mg/mL, respectively. As for anti-tumor activities, drugs were added with six different concentrations of the single free drug (free PTX) and six different concentrations of both free drugs (free PTX + free TET) at the fixed TET concentration of 5 mM. Drug-loaded nanoparticles were added with six different concentrations of the single agent (PTX-CTAB@MSN or TET-CTAB@MSN) and six different concentrations of composited agents (PTX/TETCTAB@MSN) at the fixed ratio of PTX and TET based on the respective individual IC50 values of PTX-CTAB@MSN and TETCTAB@MSN for 72 h. Dose-response curves were obtained for each free drug or agent, and for multiple dilutions of a fixed-ratio combination of the two drugs or agents. The cytotoxicity and in vitro antitumor activities were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylazolium bromide dye reduction method (the MTT assay). Briefly, MCF-7 and MCF-7/ADR cells were seeded in 96-well plates at the density of 4  103 cells/well/100 mL and 8  103 cells/well/100 mL, respectively. After the cells had been incubated for 24 h, designated wells were treated with different concentrations of free drugs or agents as described above. After 72 h of drug treatment, 20 mL of MTT (5 mg/ mL) solution were added to each well. The plate was incubated for an additional 4 h allowing viable cells to reduce the yellow tetrazolium salt (MTT) into purple blue formazan crystals, and then the medium was removed. 200 mL DMSO was added to each well to dissolve any purple formazan crystals formed. The plates were vigorously shaken before taking measurement of relative color intensity. The absorbance of each well was measured by a microplate reader (BioRad680, USA) at a test wavelength of 570 nm. The cell inhibitory rate was calculated as follows: inhibitory rate = (1-Abs570treatedcells/ Abs570controlcells)  100%. The antitumor activity was expressed as IC50 that was defined as the drug concentration required inhibiting cell growth by 50% relative to controls.

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2.10. Nuclear staining analysis by Hoechst 33342

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To address the death pattern, the MCF-7 and MCF-7/ADR cells were stained with Hoechst 33342 dye. The Hoechst 33342 dye is sensitive to chromatin and is used to assess the changes in the nuclear morphology. Briefly, MCF-7/ADR cells were seeded in 35 mm culture dishes with 3.0  105 per dish, and cultured at 37
C for 24 h. Cells were then exposed to blank MSN and CTAB@MSN with the concentration of 20 mg/mL, PTX-CTAB@MSN with the PTX

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concentration of 100 nM, TET-CTAB@MSN with the TET concentration with 22.7 nM and their composite agent PTX/TET-CTAB@MSN with 100 nM PTX and 22.7 nM TET, respectively. As the control, cells were also treated with free drugs (free PTX, free TET and free PTX + free TET) and their concentrations were ten times higher than drugs encapsulated in their corresponding drug-loaded nanoparticles. Cells were incubated for 48 h and then stained with 5 mg/mL of Hoechst 33342 for 20 min at room temperature in the dark. The Hoechst-stained nuclei were visualized by using the fluorescent microscope (OLYMPUS, IX 71, Japan).

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2.11. Cell cycle analysis

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Cell cycle distributions based on the DNA contents were determined as follows. MCF-7/ADR cells were seeded at the density of 3.0  105 cells/well in 6-well plate and allowed to attach for 24 h. Then, cells were treated with blank MSN (20 mg/mL), CTAB@MSN (20 mg/mL), free PTX (2 mM), free TET (5 mM), their combination free PTX + free TET, PTX-CTAB@MSN (200 nM PTX), TET-CTAB@MSN (45 nM TET) and their composited agent PTX/TET-CTAB@MSN, respectively. The cells were incubated for 48 h. After washing twice with PBS, cells were harvested and collected by centrifugation at 1000 rpm for 5 min, followed by fixing in ice-cold 75% ethanol under 4
C overnight. After that cells were collected by centrifugation and wash twice with PBS to remove residual ethanol. Subsequently, cells were incubated with RNase A (100 mg/mL) for 30 min at 37
C, and stained with PI solution (5 mg/mL) for 30 min followed by analyzing with a flow cytometer (FACScan, Becton Dickinson, CA). The cell distribution in phases of G1, S, and G2 were measured and results were calculated by the ModFit LT software.

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2.12. Cell apoptotic rate detected by flow cytometry

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Apoptosis was assessed by Annexin V-FITC and PI staining followed by analysis with flow cytometry. The MCF-7/ADR cells were exposed to blank MSN and CTAB@MSN with the concentration of 20 mg/mL, PTX-CTAB@MSN with the PTX concentration of 100 nM, TET-CTAB@MSN with the TET concentration with 22.7 nM and their composite agent PTX/TET-CTAB@MSN, respectively. As the control, cells were also treated with free drugs (free PTX, free TET and free PTX + free TET) and their applied concentrations were ten times higher than drugs encapsulated in their corresponding drug-loaded nanoparticles. After incubating for 48 h, MCF-7/ADR cells were trypsinized, centrifuged at 1000 rpm for 5 min, washed twice times with ice-cold PBS and re-suspended in 500 mL of binding buffer. Thereafter, 5 mL of Annexin V-FITC and 5 mL of PI were added and mixed for 15 min in the dark at the room temperature. The stained cells were analyzed using a flow cytometer.

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3. Results and discussion

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3.1. Drug loading capacities, morphology and particle size of nanoparticles

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The drug-loaded nanoparticles were prepared by self-assemble in situ drug loading approach (He et al., 2010). Drugs were firstly

solubilized in the inner core of CTAB micelles, and then the silica resource TEOS was hydrolyzed and condensed with simultaneously encapsulating CTAB micelles and drugs. In our formulations, PTX and TET were successfully encapsulated either alone or in combination. Table 1 summarized the average drug loading efficiency of nanoparticles. The PTX loading efficiency of PTXCTAB@MSN was 8.74  1.32% and the TET loading efficiency of TETCTAB@MSN was 4.23  0.67%. The amount of two drugs loaded in the composite agent PTX/TET-CTAB@MSN was adjusted according to the IC50 values of PTX-CTAB@MSN and TET-CTAB@MSN in MCF7/ADR cells. Therefore, the molar ratio of PTX to TET loaded in the composite agent was approximately regulated to 4.4/1, owing to that the IC50 values of PTX-CTAB@MSN and TET-CTAB@MSN were 228.6 nM and 52.1 nM (that will be discussed later), respectively. The morphologies of PTX-CTAB@MSN, TET-CTAB@MSN and PTX/TET-CTAB@MSN were examined by TEM as shown in Fig. 1. The images revealed that all the nanoparticles were dispersed as individual particles with a well-defined spherical structure. Besides, it was also indicated that CTAB micelles containing drugs were successfully loaded in the nanoparticles, because these nanoparticles did not exhibit typically hexagonal mesopores. The mean particle sizes of drug-loaded nanoparticles are listed in Table 1 and the particle size distribution are shown in Fig. 2. From the images, it can be observed that all the nanoparticles have narrow size distributions with low polydispersity indices (1 mm) and nanoparticles ( 0.05). After PTX was loaded in the nanoparticles (PTX-CTAB@MSN), its cytotoxicities on MCF-7/ADR cells and MCF-7 cells were all significantly improved (as shown in Fig. 7(B and D)) and the IC50 values of PTX-CTAB@MSN on sensitive cells and resistance cells were 34.4 nM and 228.6 nM, respectively. The enhancement of in vitro antitumor activity for PTX-CTAB@MSN was contributed to the following reasons. The drug-loaded nanoparticles were

Please cite this article in press as: Jia, L., et al., Multifunctional mesoporous silica nanoparticles mediated co-delivery of paclitaxel and tetrandrine for overcoming multidrug resistance. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.05.010

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Fig. 7. In vitro anti-tumor activity: (A) in vitro anti-tumor activity of free PTX and free PTX + free TET against MCF-7/ADR cells; (B) in vitro anti-tumor activity of PTXCTAB@MSN and PTX/TET-CTAB@MSN against MCF-7/ADR cells; (C) in vitro anti-tumor activity of free PTX and free PTX + free TET against MCF-7 cells; and (D) in vitro antitumor activity of PTX-CTAB@MSN and PTX/TET-CTAB@MSN against MCF-7 cells. 563 564 565 566 567 568 569 570 571 572 573 574

endocytosed by cells and PTX was delivered more efficiently into cells by nanoparticles compared with the free PTX. Besides, CTAB not only acted as the component of the nanoparticles but also was the antitumor active ingredient. Fig. 7(B and D) also illustrates the multiple drug effect obtained for MCF-7 and MCF-7/ADR cells, which were treated with PTX/TETCTAB@MSN. The combination of two drugs administered by nanoparticles generated more cell deaths than any other agents or free drugs. The IC50 values of PTX/TET-CTAB@MSN were 36.3 nM and 53.8 nM (for the concentration of PTX) for MCF-7 and MCF-7/ ADR cells, respectively. Therefore, PTX/TET-CTAB@MSN was 4-fold more effective in inhibiting the proliferation of MCF-7/ADR cells Table 2 IC50 (mean  SD) and resistance reversion index (RRI) of various agents against MCF-7 or MCF-7/ADR cells. Sample

Free PTX Free PTX + free TET PTX-CTAB@MSN PTX/TET-CTAB@MSN

MCF-7 cells

MCF-7/ADR cells

IC50 (nM)

IC50 (nM)

88.6 74.2 34.4 36.3

   

8.7 8.2 2.4 2.8

3

3.89  10  230 871.1  29.3 228.6  15.7 53.8  3.9

FR – 4.47 17.02 72.3

The reversal activity is expressed as the resistance reversion index (RRI) calculated according to the following equation: (RR) = IC50(freePTX)/IC50 (PTX formulations).

than PTX-CTAB@MSN. Besides, it was 16-fold and 72.3-fold more effective than free PTX + free TET and free PTX, respectively. So, the PTX/TET-CTAB@MSN completely reversed the resistance of MCF-7/ ADR cells to PTX and the resistance reversion index (RRI) was 72.3. The antitumor mechanism of our multifunctional nanoparticles can be concluded as follows. The PTX/TET-CTAB@MSN were taken up by MCF-7/ADR cells through endocytosis, owing to that silica particles are known to have a great affinity for the head-groups of a variety of phospholipids and the high affinity for adsorbing on cell surfaces eventually leading to endocytosis (Mornet et al., 2005; Xing et al., 2005). Subsequently, the nanoparticles were released its cargoes PTX, TET and CTAB. The intracellular TET could inhibit the efflux function of P-gp finally resulting in more and more nanoparticles entered into cells. The cargoes PTX and CTAB could perform synergistic antitumor effect on MCF-7/ADR cells to inhibit the proliferation of cells.

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Changes in morphological characteristics of the cell nucleus were analyzed by staining with Hoechst 33342 after drug treatment. The Hoechst 33342 can emit blue fluorescence and cross both the intact membrane of live cells and damaged membrane of apoptotic cells. However, it stains the condensed

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Fig. 8. Fluorescence micrographs of MCF-7/ADR cell nuclei following 48 h incubation with (A) MSN; (B) CTAB@MSN; (C) free PTX; (D) free TET; (E) free PTX + free TET; (F) PTXCTAB@MSN; (G) TET-CTAB@MSN and (H) PTX/TET-CTAB@MSN. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)

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chromatin of apoptotic cells more brightly than the looser chromatin of normal cells, and so it enables the monitoring of nuclear changes associated with apoptosis. As shown in Fig. 8, the nuclei exhibited dispersed and weak fluorescence in the control cells and the same result was obtained from cells treated with blank MSN (Fig. 8(A)) suggesting that MSN had good biocompatibility. When cells were treated with CTAB@MSN, characteristic changes of apoptosis such as chromatin condensation and the appearance of apoptotic bodies were observed in several nucleis (Fig. 8(B)). The abilities of free drugs (free PTX, free TET and free PTX + free TET) and drug-loaded nanoparticles (PTX-CTAB@MSN, TET-CTAB@MSN and PTX/TET-CTAB@MSN) to induce apoptosis or necrosis of MCF-7/ADR cells were also evaluated. Compared with free drugs (Fig. 8(C–E)), their corresponding drug-loaded nanoparticles (Fig. 8(F–H)) were all more effective on apoptosis of MCF7/ADR cells, although the concentration of free drugs was ten times higher than drugs encapsulated in drug-loaded nanoparticles implying that our obtained nanoparticles could effectively enhance the cytotoxicities of the encapsulated drugs. Among the free drugs, free PTX + free TET (Fig. 8(E)) induced most of the cell apoptosis, confirming that TET could improve the sensitivity of MCF-7/ADR cells to PTX. When PTX and TET were loaded in nanoparticles (PTX/ TET-CTAB@MSN), it promoted most of the cell apoptosis compared with all the free drugs and other formulations. It was contributed to that CTAB had synergistic antitumor effect with PTX and TET could enhance their cytotoxicities. These foundings were consistant with the conclusions of “Section 3.4”. 3.7. Cell cycle analysis To further study the induced apoptosis mechanism of our multifunctional silica nanoparticles for MCF-7/ADR cells, the

effects of free drugs and drug-loaded nanoparticle on cell cycle progression were analyzed using flow cytometry. Compared with the control group, blank MSNs showed no influence on the cell cycle progression of MCF-7/ADR cells (Fig. 9B). However, when exposed to CTAB@MSN, the cells were markedly arrested at G1 phase and the percentage of cells in G1 phase was 77.84% (Fig. 9C), which was contributed to the intracellular delivery of CTAB by MSN. It is well known that PTX could induce cell cycle arrest in the G2 phase of mitosis resulting in the restraint of cell proliferation (Belotti et al., 1996; Choi et al., 2011), whereas free PTX seemed not to affect the cell cycle progression in our study (Fig. 9D) probably due to that the concentration of PTX was too low for the drug-resistant cells. The effect of free TET on cell cycle progression of MCF-7/ADR cells was also determined. As shown in Fig. 9(E), G1 arrest was apparent after treatment with TET and the population of G1 phase increased from 50.57% to 60.34% compared with the control (p < 0.05). Many reports have also demonstrated that TET could arrest cell cycle progression at the G1 phase in other cell lines such as A549 cells and HepG2 cells to inhibit the cell proliferation and induce cell apoptosis (Kuo and Lin, 2003; Lee et al., 2002). Fig. 9(F) presents the combined effect of free PTX and free TET on cell cycle progression in the MCF-7/ADR cells. The significant G2 arrest was obtained and the population of G2 phase increased from 15.26% to 66.03% compared with the free PTX group (p < 0.01), which was contributed to that the intracellular accumulation of PTX was increased through TET inhibiting the efflux function of P-gp. When the cells were exposed to PTXCTAB@MSN, G2 arrest was showed (as shown in Fig. 9(G)). Therefore, G2 arrest induced by PTX played the vital role in inhibiting the cell proliferation for PTX-CTAB@MSN, although CTAB could also arrest cells at G1 phase as discussed above. As for TETCTAB@MSN, MCF-7/ADR cells are significantly arrested at G1 phase

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Fig. 9. Cell cycle distributions of MCF-7/ADR cells. Cells were treated with (A) control; (B) MSN; (C) CTAB@MSN; (D) free PTX; (E) free TET; (F) free PTX + free TET; (G) PTXCTAB@MSN; (H) TET-CTAB@MSN and (I) PTX/TET-CTAB@MSN. Data were obtained from experiments replicated three times and representative pictures were shown.

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with the cell population of 75.39% resulting from that both TET and CTAB could all induce G1 arrest. When cells were exposed to our composite formulation PTX/TET-CTAB@MSN, the necrosis peak was markedly displayed indicating that the composite agent might directly cause the cell death owing to arresting cell cycle progression in both G1 and G2 phases. Although the released CTAB and TET could arrest cells at G1 phase, the G2 arrest was dominant which was induced by PTX, as shown in Fig. 9(I).

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3.8. Apoptotic cell determination by Annexin V/PI staining assay

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To measure the apoptotic effect of free drugs and drug-loaded nanoparticles quantitatively, the percentage of cell apoptosis was investigated using Annexin V/PI assay based on the following mechanism. Externalization of phosphatidyl serine (PS) from the inner side to outer leaflet of the cell membrane is an important indicator of early apoptosis. Annexin V has a high affinity towards PS, therefore early apoptotic cells could be easily detected by fluorescently labeled Annexin V. Besides, PI could detect late apoptotic or necrotic cells due to its permeability through the damaged cell membranes. As shown in Fig. 10(A–C), the mean percentage of early and late apoptosis of MCF-7/ADR cells

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incubated with the control culture, blank MSN and CTAB@MSN were 8.57%, 9.25%, and 16.16%, respectively, suggesting that blank MSN was well biocompatible and CTAB@MSN induced significantly cell apoptosis (p < 0.05) duo to CTAB arresting cells in G1 phase. Compared with free PTX treatment alone, combination free PTX with free TET markedly increased the apoptosis rate from 20.85% to 25.84%. It was contributed to that TET could increase the intracellular concentration of PTX and arrest cells at G1 phase. When PTX was loaded by MSN (PTX-CTAB@MSN), the apoptosis rate was increased form 20.85% to 40.57% owing to that MSN efficiently delivered PTX and CTAB in cells, and they induced G2 and G1 arrest, respectively, to cause cell apoptosis. TETCTAB@MSN could significantly increase cytotoxicity compared with free TET (as we have discussed above) and higher apoptosis rate was also found after cells treating with TET-CTAB@MSN. The mechanism might be that TET could increase intracellular accumulation of drug-loaded nanoparticles. And besides, it could also improve the antitumor effect of CTAB by arresting cells at G1 phase. When MCF-7/ADR cells were incubated with the composite agent PTX/TET-CTAB@MSN, the apoptosis rate was markedly increased from 20.85% to 82.89% (p < 0.01). The mechanism may be as follows. The nanoparticles were taken up

Please cite this article in press as: Jia, L., et al., Multifunctional mesoporous silica nanoparticles mediated co-delivery of paclitaxel and tetrandrine for overcoming multidrug resistance. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.05.010

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Fig. 10. Flow cytometry analysis of MCF-7/ADR cells after treatment with (A) control; (B) MSN; (C) CTAB@MSN; (D) free PTX; (E) free TET; (F) free PTX + free TET; (G) PTXCTAB@MSN; (H) TET-CTAB@MSN and (I) PTX/TET-CTAB@MSN. Data were obtained from experiments replicated three times and representative pictures were shown.

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though endocytosis and then the released TET inhibited the efflux function of P-gp to increase the intracellular amount of PTX/TETCTAB@MSN. The released PTX induced G2 arrest, besides TET and CTAB arrested cells at G1 phase finally leading to cell apoptosis enhancement.

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4. Conclusion

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In the present study, the Chinese traditional medicine “tetrandrine” and conventional antitumor drug “paclitaxel” were firstly co-delivered by the multifunctional mesoporous nanoparticle to overcome multidrug resistance of MCF-7/ADR cells. The obtained PTX/TET-CTAB@MSN possessed pH-responsive release property contributing to CTAB as the “switch” to control drugs more easily released at the acid environment of tumors and hence reduced side effects and toxicities to normal tissues. The cargoes TET, as the

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chemosensitizer, could inhibit the efflux function of P-gp to increase the intracellular accumulation of drugs or nanoparticles. Besides, our studies on the mechanism demonstrated that TET could also arrest MCF-7/ADR cells at G1 phase resulting in enhancement the antitumor activities of PTX and CTAB. In the antitumor activity test, the PTX/TET-CTAB@MSN significantly inhibited the proliferation of the drug-resistant cells and completely reversed the resistance to paclitaxel. Therefore, our study provided a promising approach to overcome multidrug resistance of tumors.

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Acknowledgements

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The financial supports of this work are the National Natural Science Fund Project of China (No. 81402868) and Natural Science Foundation Program of Shandong Province (No. BS2014YY007).

Please cite this article in press as: Jia, L., et al., Multifunctional mesoporous silica nanoparticles mediated co-delivery of paclitaxel and tetrandrine for overcoming multidrug resistance. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.05.010

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