European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

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Research paper

Bufalin loaded biotinylated chitosan nanoparticles: An efficient drug delivery system for targeted chemotherapy against breast carcinoma Xin Tian a, Hongzhuan Yin b, Shichen Zhang c, Ying Luo d, Kai Xu e, Ping Ma a, Chengguang Sui a, Fandong Meng a, Yunpeng Liu d,⇑, Youhong Jiang a,⇑, Jun Fang f,g,1 a

Molecular Oncology Department of Cancer Research Institute, the First Hospital of China Medical University, Shenyang, PR China Department of General Surgery, Shengjing Hospital of China Medical University, Shenyang, PR China Department of Child & Maternal Health, Anhui Medical University, Hefei, PR China d Department of Medical Oncology, the First Hospital of China Medical University, Shenyang, PR China e School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, PR China f Laboratory of Microbiology and Oncology, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan g Department of Toxicology, Anhui Medical University, Hefei, PR China b c

a r t i c l e

i n f o

Article history: Received 11 December 2013 Accepted in revised form 11 May 2014 Available online xxxx Keywords: Bufalin Chitosan Biotin Tumor targeting EPR effect Apoptosis

a b s t r a c t Bufalin is a traditional oriental medicine which is known to induce apoptosis in many tumor cells, and it is thus considered as a new anticancer therapeutic. By now, most of the studies of bufalin are in vitro, however in vivo evaluations of its therapeutic efficacy are less and are in great demand for its development toward anticancer drug. One of the problems probably hampering the development of bufalin is the lack of tumor selectivity, which may reduce the therapeutic effect as well as showing side effects. To overcome this drawback, in this study, we designed a tumor-targeted drug delivery system of bufalin based on enhanced permeability and retention (EPR) effect, by using biotinylated chitosan, resulting in bufalin encapsulating nanoparticles (Bu-BCS-NPs) with mean hydrodynamic size of 171.6 nm, as evidenced by dynamic light scattering and transmission electron microscope. Bu-BCS-NPs showed a relative slow and almost linear release of bufalin, and about 36.8% of bufalin was released in 24 h when dissolved in sodium phosphate buffer. Compared to native bufalin, Bu-BCS-NPs exhibited a stronger cytotoxicity against breast cancer MCF-7 cells (IC50 of 0.582 lg/ml vs 1.896 lg/ml of native bufalin). Similar results were also obtained in intracellular reactive oxygen species production, apoptosis induction, and decrease in mitochondria membrane potential. These results may contribute to the rapid intracellular uptake of nanoparticles, partly benefiting from the highly expressed biotin receptors in tumor cells. In vivo studies using MCF-7 tumor models in nude mice confirmed the remarkable therapeutic effect of Bu-BCS-NPs. These findings suggest the potential of Bu-BCS-NPs as an anticancer drug with tumor targeting property. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Targeted therapy is recently among the most attractive strategies for cancer, the major cause of death in most advanced Abbreviations: Bu, bufalin; CS, chitosan; BCS, biotinylated chitosan; NPs, nanoparticles; EPR effect, enhanced permeability and retention effect; ROS, reactive oxygen species. ⇑ Corresponding authors. Molecular Oncology Department of Cancer Research Institute, the First Hospital of China Medical University, Shenyang 110001, PR China. Tel.: +86 24 83282354; fax: +86 24 83282473 (Y. Jiang). Department of Medical Oncology, the First Hospital of China Medical University, Shenyang 110001, PR China. Tel.: +86 24 83282312; fax: +86 24 83282543 (Y. Liu). E-mail addresses: [email protected] (Y. Liu), cmuliuyunpeng@hotmail. com (Y. Jiang), [email protected] (J. Fang). 1 Tel.: +81 96 326 4137; fax: +81 96 326 5048.

countries in the world. Conventional cancer chemotherapy, which usually utilizes small molecular drugs, is far from successful, mostly due to the lack of tumor selectivity, or the so-called doselimiting toxicity, resulting severe adverse effects that limits usage. To overcome these drawbacks, targeted anticancer therapy is aiming at a more tumor-selective anticancer effect with less system side effects. One direction of targeted anticancer therapy is the so-called molecular target therapy, which focuses on specific kinases or receptors that are over-expressed in cancer cells or tissues. However, considering the intrinsic genetic diversity of human solid tumors [1,2], namely high frequency of occurrence of mutant genes, redundant genetic and molecular or metabolic pathways, single gene or receptor concept of molecular target therapy may

http://dx.doi.org/10.1016/j.ejpb.2014.05.010 0939-6411/Ó 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: X. Tian et al., Bufalin loaded biotinylated chitosan nanoparticles: An efficient drug delivery system for targeted chemotherapy against breast carcinoma, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.05.010

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X. Tian et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

be invalidated. In fact, recent clinical results of many molecular target drugs have not been successful [3,4]. Another approach that is attracting more and more attentions, is a more general tumor targeting according to the unique anatomical and pathophysiological nature of solid tumor tissues. Namely, most solid tumors have blood vessels with defective architecture and usually produce extensive amounts of various vascular permeability factors, so that macromolecules with molecular weight larger than 40–50 kDa will extravasate from tumor blood vessels and accumulate selectively in tumor tissues, whereas they could not cross normal blood vessels which will result in less side effects. This unique phenomenon was first reported my Matsumura and Maeda in 1986, and was coined as enhanced permeability and retention (EPR) effect [5]. The EPR effect is thus considered to be a landmark principle in targeted cancer chemotherapy and is becoming an increasingly promising paradigm and ‘‘gold standard’’ for anticancer drug development [6–10]. To date, some macromolecular drugs are used in clinic, for example, Doxil that is a liposome formulation of doxorubicin used for treatment of Kaposi sarcoma and other cancers; and more polymeric or micellar drugs are in clinical stage development [9–11]. Along this line, recently we focused on biotin modified chitosan nanoparticles (BCS-NPs) as a drug carrier for tumor targeting. Chitosan is a linear polysaccharide with a number of commercial and possible biomedical uses. Because of its biocompatibility and biodegradability, chitosan has been widely used as polymer materials for the modification and delivery of many anticancer drugs, i.e., chitosan nanoparticles (CS-NPs) [12–15]. Biotin is a watersoluble vitamin, having essential roles involving cell growth, signal transduction and many other cellular functions, it is internalized into the cells through binding to the sodium dependent multivitamin transporter (SMVT) in the cell surface [16,17]. Importantly, many tumor cells highly express this transporter to meet the demand of biotin for rapid tumor growth, and biotinylation is thus a reasonable strategy to enhance the binding/affinity of macromolecular drugs to tumor cells, leading to increasingly effective antitumor therapy [18–21]. It has been reported that biotinylated nanoparticles selectively bound to breast cancer MCF-7 cells resulting in higher intracellular uptake than non-modified nanoparticles [12,22,23]. Therefore, utilization of BCS-NPs would exhibit double tumor-targeting effect, namely, it will selectively accumulate in tumor tissues by EPR effect, and then targetedly bind to and internalized into tumor cells that highly express biotin receptors. Based on these notions, in this study, we prepared BCS-NPs with the use of bufalin as the antitumor entity. Bufalin is a traditional oriental medicine that is the major digoxin-like immunoreactive component of Chan Su, obtained from the skin and parotid venom glands of toads [24]. Bufalin is a cardioactive C-24 steroid that exhibits a variety of biological activities, especially regulating cardiovascular functions [24,25]. Recently it is also known to induce cell cycle arrest and apoptosis in many cancer cells [24,26], and is thus considered a candidate drug for cancer chemotherapy. However, like many conventional anticancer drugs, the small molecular nature of bufalin hampered its development and application, especially the structural similarity of bufalin to digoxin may cause severe toxic effect if indiscriminately distributed in the body. Accordingly, the aim of this study was to investigate the efficacy and possibility of BCS-NPs as the drug delivery system for bufalin to increase the tumor selectivity as well as decrease toxic effects. The preparation, physiochemical characterization of bufalin loaded BCS-NPs (Bu-BCS-NPs) are described, the in vitro as well as in vivo antitumor effect are then examined using a breast cancer MCF-7 cell line and its mice xenograft model, by comparison with unmodified native bufalin.

2. Materials and methods 2.1. Chemicals Bufalin and 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chitosan (CS) with a mean molecular weight of about 1  106 Da was from AK Biotech Ltd. (Jinan, China). The deacetylation degree of CS which we used is 91.5%, and the polydispersity index (Mw/ Mn) is 1.36 tested by the gel permeation chromatography (GPC) method, as provided by the manufacturer. Sulfosuccinimidobiotin (sulfo-NHS-biotin) were purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA). All other chemicals and reagents were from commercial sources unless otherwise described. 2.2. Cell culture Human breast cancer cells MCF-7 were kindly provided by Department of Pharmacology, Shenyang Pharmaceutical University. The cells were cultured in RPMI-1640 medium (Sigma) with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37 °C in an atmosphere of 5% CO2/95% air. 2.3. Synthesis of biotinylated chitosan Biotinylated CS (BCS) was synthesized according to a method by Yao et al. with some modifications [27]. In brief, 100 mg CS was dissolved in 5 ml of 10% HCl and the pH was adjusted to 6.5. To this solution, 5 mg sulfo-NHS-biotin (0.83 ml) was added dropwise and stirring for 24 h at room temperature. Afterward, the resultant was subjected to dialysis with a membrane of 8000–10000 Da molecular cut-off, against deionized water for 24 h with 3-change of water, followed by lyophilization. According to this protocol, the binding rate of biotin was quantified to be about 2.5 mol/mol CS by using a biotin assay kit (Pierce Biotechnology, Inc.). In some experiments, to obtain an FITC labeled BCS (FITC-BCS), 100 mg of BCS was dissolved in 5 ml of 0.01 M phosphate-buffered 0.15 M saline (PBS; pH 7.4), to which 0.1 ml of FITC (5 mg/ml in acetone) was added and reacted for 2 h at room temperature avoiding the light. The resulted FITC-BCS was collected after centrifugation (150,000 rpm, 30 min) and lyophilized. 2.4. Preparation of bufalin-BCS nanoparticles (Bu-BCS-NPs) The Bu-BCS-NPs were prepared by solvent-dialysis method. Briefly, 10 mg bufalin and 50 mg BCS were dissolved in dimethyl sulfoxide, and the solution was subjected to sonication for 5 min. Then, by the use of a dialysis membrane with molecular cut-off of 3500 Da, the solution was dialyzed against deinonized water for 3 days, with the change of water every 6 h. After dialysis, the solution was collected and subjected to sonication for 15 min, after which it was filtered with a syringe filter with a 0.45 lm pore size hydrophilic PVDF membrane (Millex-HV Filter, 0.45 lm, Merck Millipore, Billerica, MA, USA). The filtrate was lyophilized to obtain Bu-BCS-NPs. In some experiments, nanoparticles without biotinylation (BuCS-NPs) and FITC labeled Bu-BCS-NPs were prepared by the same protocol, using CS without biotin modification and FITC-BCS respectively. 2.5. Characterization of Bu-BCS-NPs 2.5.1. Determination of encapsulation efficiency (EE) and drug loading (DL) The EE and DL of bufalin were determined by HPLC method. Generally, 10 mg of Bu-BCS-NPs was first suspended in 1 ml of

Please cite this article in press as: X. Tian et al., Bufalin loaded biotinylated chitosan nanoparticles: An efficient drug delivery system for targeted chemotherapy against breast carcinoma, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.05.010

X. Tian et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

50 mM sodium phosphate buffer (pH 7.4), and 5 ml N,N-dimethylformamide was added. The completely dissolved solution was then obtained after sonication, and 20 ll was subjected to a Shimadzu LC-10AT series HPLC system (Shimadzu Co., Kyoto, Japan) with a DiamonsilTM C18 column (10 lm, 300 mm  4.6 mm, Dikma Co., Beijing, P.R. China). The mobile phase was acetonitrile/methanol/ water (10:60:30, v/v) and the flow-rate was 1.0 ml/min, with a detection wavelength of 298 nm. The column temperature was kept at 30 °C during the experiments. The bufalin content in the NPs was thus calculated by referring the bufalin standard. The EE and DL were thus calculated by the following formulas: EE = total amount of bufalin in obtained NPs/bufalin feeding for the preparation; DL = total amount of bufalin in obtained NPs/total amount of obtained NPs. 2.5.2. Dynamic light scattering and zeta potential Bu-BCS-NPs were dissolved in PBS at 1 mg/ml and was filtered through a 0.45 lm filter. The particle size and surface charge (zeta potential) were measured by using a light-scattering instrument (Zetasizer Nano; Malvern Instruments Ltd., Worcestershire, UK). 2.5.3. Transmission electron microscopy (TEM) A drop of Bu-BCS-NPs solution (0.05 mg/ml) was applied to a copper grid coated with carbon film and air-dried. The micelle image and the size of Bu-BCS-NPs were analyzed by means of a transmission electron microscope (Tecnai F20; FEI, Hillsboro, OR, USA). 2.5.4. Release rate of bufalin from Bu-BCS-NPs The release of free drug (bufalin) from Bu-BCS-NPs was examined in vitro. Namely, 10 mg of Bu-BCS-NPs was dissolved in 1 ml 0.1 M sodium phosphate buffer (pH 7.4) and placed in sealed dialysis tube (Mw cut-off 1000 Da; Spectrapor, Spectrum Laboratories Inc., San Diego, CA). The dialysis tubes were submerged in 100 ml pH 7.4 sodium phosphate buffer containing 0.5% Tween 80. The tubes were then incubated for several hours at 37 °C in the dark with stirring. The bufalin released from the dialysis bags was collected at predetermined time intervals (i.e., 0.5, 1, 2, 4, 8, 12, 24 h), and quantified by HPLC as described above. Similar experiment was carried out by use of free bufalin that dissolved in ethanol. 2.6. In vitro cytotoxicity of Bu-BCS-NPs MCF-7 cells were plated at 5000 cells/well in a 96-well plate (Nunc A/S, Roskilde, Denmark). After 24 h preincubation, the medium was changed to fresh medium containing different concentrations of Bu-BCS-NPs, Bu-CS-NPs or free bufalin, and incubated for 48 h. The cell viability was then determined by using the MTT assay (Dojindo Laboratories, Kumamoto, Japan). 2.7. Intracellular uptake of Bu-BCS-NPs MCF-7 cells (2.5  104) were cultured in 6-well plate (Nunc A/S) in RPMI-1640 medium with 10% FCS without phenol red. After 24 h preincubation, 0.1 ml of Hochest 33343 (dissolved in medium) was added and indicated at 37 °C for 1 h. Then, the medium was removed and the cells were washed twice by PBS, and incubated in fresh medium containing FITC-Bu-BCS-NPs (1 lg/ml) for 8 h. The intracellular uptake of Bu-BCS-NPs was then analyzed by the fluorescence of FITC using a fluorescence Microscope (Olympus, Japan). The intracellular uptake was also quantified by bufalin encapsulated in the cells. Namely, MCF-7 cells were placed in 12-well plate (5  105 cells/well). After 24 h preincubation, cells were treated with Bu-BCS-NPs, Bu-CS-NPs (dissolved in PBS) or free bufalin (dissolved in ethanol) for indicated time at the concentrations of

3

100 ng/ml (bufalin equivalent). Then, cells were harvested by trypsinization and washed with PBS thrice at 4 °C. After sonication (30 W, 30 s, UP50H homogenizer, Dr. Hielscher GmbH, Teltow, Germany) in ethanol in ice, cells were centrifuged to collect supernatant containing bufalin, which was quantified by HPLC as described above. 2.8. Detection of intracellular reactive oxygen species (ROS), mitochondria membrane potential (DW(m)) and apoptosis MCF-7 cells were seeded in 12-well plates (105 cells/well). After 24 h preincubation, cells were treated with Bu-BCS-NPs, Bu-CS-NPs or free bufalin for 24 h at the concentrations of 100 ng/ml (bufalin equivalent). Then, 10 lM DCDHF-diacetate was added, and the cells were cultured for an additional 30 min. The amount of intracellular ROS was quantitated as a function of fluorescence intensity of dichlorofluorescein that resulted from the reaction of DCDHF with ROS inside cells, measured by flow cytometry (BD FACSCalibur 3A; Becton Dickinson, San Jose, CA). For detecting DW(m), 2 lM JC-1 dye (Life Technologies Ltd., Carlsbad, CA, USA) [28] was added after the same treatment protocol as described above, and incubated for 15 min. Analyses were carried out by flow cytometry (BD FACSCalibur 3A) according to the manufacturer’s instructions. The change of DW(m) was also investigated by using Mitotracker Red (Life Technologies Ltd.) that accumulates in mitochondria dependent on membrane potential, which was detected by confocal microscope (FV1000s, Olympus, Japan) after 30 min incubation at 100 nM. Induction of apoptosis was determined by a flow cytometric assay with Annexin V-FITC. In brief, after above-described treatment, the cells were harvested by use of a rubber policeman, then subjected to staining with the Annexin V-FITC kit and propidium iodide. The number of apoptotic cells was determined by flow cytometry (BD FACSCalibur 3A). 2.9. Western blot analysis of apoptosis related molecules To further examine apoptosis induction involved in the Bu-BCSNPs treatment, we investigated the activation of four apoptosis related molecules, i.e., Bcl-2, Bax, survivin and caspase, using western blot analysis. MCF-7 cells were treated by Bu-BCS-NPs, Bu-CS-NPs or free bufalin as the same protocol as described in Section 2.8, after which the cells were harvested and centrifuged at 3000 rpm for 5 min. The cell pellets were then homogenized with ice-cold homogenate buffer (20 mM Tris–HCl, pH 7.4 plus 3 mg/ ml PMSF and 3 mM EDTA), and centrifuged at 10,000 rpm for 15 min at 4 °C. The supernatant thus obtained was loaded into 20 mM Tris–HCl, pH 7.4 plus 1% Triton X-100. The proteins in the supernatant were quantitated according to the Lowry method, and were separated by electrophoresis with 12% SDS-polyacrylamide gels and transferred to immobilon polyvinylidene difluoride membranes (Millipore Co. Ltd., Bedford, MA). The antibodies against Bcl-2, Bax, survivin, caspase and cleaved (activated) caspase were rabbit polyclonal antibodies (Santa Cruz Biotech. Inc. Dallas, TX, USA) and were used according to the manufacturer’s instructions, followed by a subsequent second antibody of goat anti-rat IgG (Beijing Zhongshan Biotech Co., Beijing, China). The protein band that reacted immunologically with the antibody was visualized by using the enhanced chemiluminescence system (ECL; Amersham Biosciences, Buck, UK). 2.10. In vivo antitumor effect of Bu-BCS-NPs Female Balb/c nude mice, 4 weeks old and weighing 20 g were obtained from Animal Facility of China Medical University (Shenyang, P.R. China). All animals were maintained under SPF

Please cite this article in press as: X. Tian et al., Bufalin loaded biotinylated chitosan nanoparticles: An efficient drug delivery system for targeted chemotherapy against breast carcinoma, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.05.010

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conditions and were fed sterilized water and murine chow ad libitum. All experiments were carried out according to the Guidelines of the Laboratory Protocol of Animal Handling, China Medical University. Tumor model was established by implanting MCF-7 cells (1  107 cells) s.c. in the right axilla skin of mice. The in vivo antitumor study was performed on days 7–10 after tumor inoculation, when tumors were 4–5 mm in diameter and had no necrotic region. Bu-BCS-NPs (1 mg/kg bufalin equivalent, 0.2 ml in PBS) or free bufalin (1 mg/kg, 0.2 ml in ethanol) was administered i.p., every two days for 20 days with the total dose of 10 mg/kg, and in a separate group, Bu-BCS-NPs were injected i.v. (5 mg/kg bufalin equivalent, 0.1 ml in PBS) at days 1 and 3 of the treatment with the same total dose of 10 mg/kg. In control experiments, mice received physiological saline (0.2 ml, i.p.). The tumor volume and body weight of the mice were measured every 2–3 days during the period of investigation. Values for the tumor volume (V) were determined by measuring the longitudinal cross section (L) and the transverse section (W) and then applying the formula V = (L  W2)/2.

with two experiments were analyzed by Mann–Whitney U-test. The difference was considered statistically significant at P < 0.05. 3. Results and discussions 3.1. Synthesis and characterization of Bu-BCS-NPs Bu-BCS-NPs were prepared by solvent-dialysis method, replacing the organic solvent to water gradually, during which NPs formed probably with the core of hydrophobic bufalin and surrounded by CS as the outer surface facing water (Fig. 1A). The resultant Bu-BCS-NPs revealed a spherical appearance, most of which showed diameters of 50–200 nm (Fig. 1B). And we observed a single-peak distribution of Bu-BCS-NPs by dynamic light scattering, with a hydrodynamic particle size of 165.3 ± 71 nm in aqueous solution (Fig. 1C). Moreover, the zeta potential of Bu-BCS-NPs was +16.5 mV, which is slightly higher than common chitosan NPs (i.e., +13 mV), probably due to the biotinylation in the surface of chitosan NPs (Fig. 1A). In addition, during the preparation of CS nanoparticles, a filtration using a syringe filter with a 0.45 lm pore size was carried out which was used for sterilization. The particle size before filtration was 175.6 ± 61.8 nm, indicating the filtration did not affect significantly the particle size.

2.11. Statistical analysis All data are presented as means ± SD. Data were analyzed by one-way ANOVA followed by the Bonferroni t-test. Some studies

A

CS

Bu-BCS-NPs

CS

Biotin Bu Bu

C Intensity (%)

B

20 15 10

5 500nm

0 10

100

1000

10000

Size (nm)

D

Particle size

171.6

31.3 nm

Zeta potential

+ 16.5 mV

Encapsulation efficiency (EE)

~ 77.4%

Bufalin loading (DL)

~ 13 wt/wt %

Fig. 1. Diagrammatic illustration (A) and characterization of Bu-BCS-NPs (B–D). TEM of Bu-BCS-NPs is shown in (B). The hydrodynamic size of Bu-BCS-NPs in PBS determined by dynamic light scattering (DLS) is shown in (C), and the physiochemical characteristics of Bu-BCS-NPs are summarized in (D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: X. Tian et al., Bufalin loaded biotinylated chitosan nanoparticles: An efficient drug delivery system for targeted chemotherapy against breast carcinoma, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.05.010

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improved water solubility (>20 mg/ml in PBS) compared to native bufalin, which makes it possible for systemic administration thus increasing its applicability.

120

Bu-BCS-NPs 100

Bufalin

3.2. Release of bufalin from Bu-BCS-NPs

% Release

80 60 40 20 0 0

4

8

12

16

20

24

Time (h) Fig. 2. Release rate of bufalin from Bu-BCS-NPs. Bu-BCS-NPs were dissolved in 1 ml PBS and placed in sealed dialysis bags, and were submerged in 0.1 M sodium phosphate buffer containing 0.5% Tween 80, pH 7.4. The bufalin diffused across the dialysis bags were collected at indicated time intervals and its amount quantified by HPLC referring to free bufalin. See text for details.

According to this method, 3 lots of Bu-BCS-NPs were prepared. Based on the results of HPLC, the EE of bufalin in the NPs were 76.6%, 80.2% and 75.4% respectively (77.5 ± 2.5%), and the loadings of bufalin in the NPs were 12.8%, 13.4% and 12.6% respectively (12.9 ± 0.4%, w/w), suggesting a high reliability of this method. Furthermore, the drug loading efficiency of non-filtrated particles was about 13.6%, and no significant change was observed between NPs before and after the filtration. The resultant Bu-BCS-NPs showed

90

120

B Bu-CS-NPs

80

C

100

Bu-BCS-NPs

70

Bufalin 60

% Survival

Bufalin (ng/ml)

Merge

FITC-Bu-BCS-NPs

Hoechst 33342

A

Release of active drugs is a critical issue involved in the efficacy and application of macromolecular drugs including micelles, nanoparticles, drug–polymer conjugates as well as liposome. Macromolecular drugs commonly show prolonged circulation time and selective tumor accumulation benefiting from the EPR effect. Nevertheless, release of active principles is also necessary, which will greatly affect the therapeutic effect. Namely, too slow a release results in insufficient concentrations of active drugs, whereas too rapid would lead to a high concentration of free drug in circulation but no drug accumulation in the tumor. Both results will thus lead to a considerably lower therapeutic effect and the latter may also induces undesired systemic toxicity. Therefore, study of drug release kinetics is an indispensable evaluation for nanoparticles, micelles and other macromolecular drugs. We thus investigated the release rate of Bu-BCS-NPs in a physiological solution. As shown in Fig. 2, parental bufalin diffused freely and crossed the dialysis membrane rapidly, i.e. almost all bufalin diffused across the dialysis membrane within 2–4 h, whereas Bu-BCS-NPs in PBS solution manifested a slow and sustained release of bufalin which could diffuse across the dialysis membrane; a nearly linear release rate of about 1.5% per hour was found and less than 40% of bufalin was released within 24 h. Consideration of the EPR effect which could be observed in hours after drug administration and lasted for days [6], the release rate of Bu-BCS-NPs is reasonable. Namely, the slow release rate will

50 40

80 60 40

30

Bu-BCS-NPs

20 20 10

Bufalin Bu-CS-NPs

0 30

60

120

Time (min)

240

480

0 0.001

0.01

0.1

1

10

Drug concentration (µg/ml)

Fig. 3. Intracellular uptake and cytotoxicity of bufalin and its nanoparticles. (A) Fluorescence microscopic analysis of MCF-7 cells treated with FITC-Bu-BCS-NPs at 8 h after incubation. Substantial green fluorescence (FITC-Bu-BCS-NPs) was observed in the cells. (B) Intracellular uptake of free bufalin, Bu-CS-NPs, and Bu-BCS-NPs at different time after incubation, as measured by using HPLC analysis. Values are means ± S.E. (n = 4). (C) Cytotoxicity of bufalin, Bu-BCS-NPs and Bu-CS-NPs as measured by MTT assay. Values are means ± S.E. (n = 8). P < 0.05; P < 0.01. See text for details.

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not induce the abrupt increase in circulating concentration of the free drug, thus little severe toxic effect will be triggered; meanwhile, the sustained release will gradually increase the tumor concentration of the active drug while accumulating and retaining in the tumor (EPR effect), finally resulting in high therapeutic effect as described below (in vivo antitumor effect).

3.3. Intracellular uptake and in vitro cytotoxicity of Bu-BCS-NPs Intracellular uptake is another key step of macromolecular drugs to achieve successful therapeutic effect because most antitumor agents need to access and react to cellular components, such as DNA, mitochondria. However, low intracellular uptake is often found for macromolecular drugs accompanying with lower cytotoxicity, for example, PEG dilemma is known as a phenomenon of pegylated drugs usually showing low intracellular uptake [29,30]. To overcome this drawback, some strategies were developed, one of which is to control the release of active drugs selectively in tumor environments by using tumor milieu-responsive biodegradable linkage, for example hydrazone bond (cleaved in

A

acidic tumor pH, i.e., 6.7) [31], and cathepsin B-cleavable peptide linkage glycylphenylalanylleucylglycine (GFLG) [32]. Another widely used strategy is aiming at the special molecules or receptors in tumor cells using, for example, transferrin, folate, integrin receptors, epidermal growth factor, antibodies, glycoprotein, etc., namely active targeting [33–35]. Along this line, in this study, to further enhance the tumor targeting efficacy, an active targeting moiety, biotin, was used to modify CS, because it is known that many tumor cells highly expressed biotin receptors [18–21]. Biotinylation thus becomes a useful tool of targeting tumor beyond EPR effect for more augmented tumor accumulation and cell internalization. As expected, biotinylated CS nanoparticles (Bu-BCS-NPs) exhibited a substantial intracellular uptake in MCF-7 cells using fluorescence microscopy (Fig. 3A). This internalization was significantly more rapid and higher than that of non-modified CS nanoparticles (Bu-CS-NPs) (Fig. 3B). More importantly, it was also higher than that of free bufalin after 8 h of treatment (Fig. 3B). These findings suggested that Bu-BCS-NPs are actively internalized via transporters in cell membrane resulting in the time-dependent increase in uptake, whereas free bufalin entered into cells via free diffusion that showed saturation of uptake.

B

Control

E 1400

Bu-BCS-NPs

Bufalin 6.47%

1.8%

Mean FL intensity

1200

Bufalin Bu-CS-NPs Control

1000 4.64%

3.86% 800 600

Bu-BCS-NPs

Bu-CS-NPs

400

2.69%

11.25%

6.18%

12.46%

200 0

35 % Apoptotic cells

C

30 25 20 15 10 5 0

Control

FL2 (JC-1 aggregates, red FL)

D

Bufalin

Bu-CS-NPs

Control

Bu-BCS-NPs

F Control 94.25%

Bufalin 84.32%

Bu-CS-NPs 87.63%

Bu-BCS-NPs

Con CS-NPs

Bufalin

Bu

Bu-CS-NPs Bu-BCS-NPs

BCS-NPs

Bcl-2

72.77%

Bax Survivin 7.45%

15.58%

12.37%

FL1 (JC-1 monomers, green FL)

27.12%

Caspase-3 Cleaved Caspase-3 GAPDH

Fig. 4. Increased intracellular ROS, loss of DW(m) and apoptosis induced by Bu-BCS-NPs. MCF-7 cells were seeded in 12-well plates (105 cells/well). After 24 h preincubation, cells were treated with Bu-BCS-NPs, Bu-CS-NPs or free bufalin for 24 h at the concentrations of 100 ng/ml (bufalin equivalent). Intracellular ROS was detected by DCDHF with an FACS assay (A) and the quantitated data is shown in (B). DW(m) was examined by Mitotracker Red staining using confocal fluorescence microscopic analysis in which less fluorescence indicates the dysfunction of mitochondria or loss of DW(m) (C), and by JC-1 staining using an FACS analysis in which decrease in JC-1 aggregates (FL2) and increase in JC-1 monomers (FL1) indicates the loss of DW(m) (D). Apoptosis was detected by an FACS assay with annexin-V (E), and the expressions of apoptosis-related molecules Bcl-2, Bax, survivin and caspase-3 were measured by western blot (F). Con, control; Bu, bufalin; CS-NPs, Bu-CS-NPs; BCS-NPs, Bu-BCS-NPs. Data are means ± S.E. (n = 4). P < 0.05; P < 0.01. See text for details.

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Because of the higher intracellular uptake, the stronger cytotoxicity of Bu-BCS-NPs compared to free bufalin was thus expected, and a MTT assay was carried out. As shown in Fig. 3C, Bu-BCS-NPs exhibited a much higher cytotoxicity to MCF-7 cells, with an IC50 of 58.2 ng/ml compared to that of free bufalin (189.6 ng/ml), a >3time increased antitumor effect. Moreover, Bu-CS-NPs showed lower cytotoxicity than free bufalin and Bu-BCS-NPs (Fig. 3C). These results well coincided with the findings of intracellular uptake, suggesting that the effect of bufalin NPs is at least partly dependent on the internalization into the cells, and thus biotinylation ensure the increased and targeted antitumor efficacy of Bu-BCS-NPs. 3.4. Increased intracellular ROS, loss of DW(m) and apoptosis induced by Bu-BCS-NPs It was reported that bufalin triggered apoptosis via induction of ROS, loss of DW(m) in many tumor cells including melanoma cells, lung cancer cells and bladder cancer cells [36–38], we thus investigated the effect of Bu-BCS-NPs on intracellular ROS, DW(m) and apoptosis in MCF-7 cells, and compared with free bufalin. By the use of an intracellular ROS probe DCDHF, we found a significantly increased ROS in MCF-7 cells treated by bufalin (Fig. 4A and B). Bu-BCS-NPs induced a further increase in intracellular ROS which is about 2.5 times that of free bufalin (Fig. 4B). Consistent with these findings, loss of mitochondria membrane potential that is considered the early phenomenon of apoptosis was observed, both by using Mitotracker (Fig. 4C) and by using commonly-used DW(m) probe JC-1 (Fig. 4D). In both cases, Bu-BCS-NPs triggered more mitochondria dysfunction than free bufalin, which related to the significantly increased numbers of apoptotic cells as showed in Fig. 4E. Induction of apoptosis was also verified by the expression of anti-apoptotic molecules Bcl-2 and survivin whose expression obviously down-regulated after Bu-BCS-NPs treatment, and pro-apoptotic molecules Bax and caspase-3, especially cleaved caspase-3 that highly up-regulated by Bu-BCS-NPs (Fig. 4F). These findings supported previously reported antitumor mechanisms of bufalin, and suggested that the active entity of Bu-BCS-NPs is bufalin released from nanoparticles after Bu-BCS-NPs were internalized and disrupted in lysosome. In fact, Bu-CS-NPs which are without biotinylation and showed lower intracellular uptake than 6000

free bufalin and Bu-BCS-NPs (Fig. 3B), also exhibited lower effects on induction of ROS and apoptosis (Fig. 4); moreover, empty nanoparticles without bufalin did not show apparent cytotoxicity to MCF-7 upto the concentration of 1 mg/ml (data not shown). Regarding the antitumor mechanisms, though ample evidences indicated apoptosis is the major pathological change of bufalininduced cell death [26], autophagy-mediated cell death was recently reported to be induced by bufalin via ROS, JNK or AKT/ mTOR dependent pathway [39,40]. In view of the apoptosis induction pathway, ROS and loss of DW(m) were known the major triggers [36–38] that were supported by the findings in the present study (Fig. 4), and many signal transduction pathways were proven to be involved, for example, NF-related apoptosis-inducing ligand (TRAIL) [26]. Understanding the molecular pathways will greatly help to open the door for the development of bufalin as an anticancer drug. However, most studies described above were in vitro study, in vivo evaluation of the actions of bufalin is thus needed. This might be achieved by use of suitable drug delivery system, such as BCS-NPs as showed in this study, which warranted further investigations. In this study, by using this drug delivery platform, we then examined the in vivo antitumor effect of Bu-BCS-NPs. 3.5. In vivo antitumor effect of Bu-BCS-NPs The in vivo study was carried out using a human breast cancer MCF-7 xenograft model in nude mice, and the effect of Bu-BCS-NPs was compared to free bufalin. Because bufalin could not dissolved in physiological solution, it was applied via an i.p. route with multiple administration (10 injections), which is commonly used treatment protocol for bufalin to maintain blood concentration and to achieve therapeutic effect. For comparison, in some experiments Bu-BCS-NPs were administered both by the same i.p. protocol. Moreover, because of the EPR effect based merits i.e., prolonged circulation time and tumor targeting property, macromolecular drugs (e.g. nanoparticles) can achieve satisfied therapeutic effect with less numbers of administration by i.v. injection, thus we also administered Bu-BCS-NPs by i.v. route with only two injection in some experiments. However, the total amount of drug (bufalin equivalent) used in each treatment group was same, i.e., 10 mg/kg. 35

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Fig. 5. In vivo antitumor effect of Bu-BCS-NPs. MCF-7 cells (1  107 cells) was inoculated s.c. in the axilla skin of Balb/c nude mice. At days 7–10 when tumors were 4–5 mm in diameter, Bu-BCS-NPs (1 mg/kg bufalin equivalent) or free bufalin (1 mg/kg) was administered i.p., every two days for 20 days (total dose of 10 mg/kg). In a separate group, Bu-BCS-NPs were injected i.v. (5 mg/kg bufalin equivalent) at day 1 and 3 of the treatment (total dose of 10 mg/kg). In control experiments, mice received physiological saline (0.2 ml, i.p.). The antitumor effect of each treatment is shown in (A), and the change of body weight during the treatments is shown in (B). Data are means ± S.E. (n = 6).  P < 0.05; P < 0.01. See text for details.

Please cite this article in press as: X. Tian et al., Bufalin loaded biotinylated chitosan nanoparticles: An efficient drug delivery system for targeted chemotherapy against breast carcinoma, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.05.010

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As showed in Fig. 5A, suppression of tumor growth was achieved by free bufalin. However, accompanying with this antitumor effect, a significantly decreased body weight was found (Fig. 5B), suggesting the toxic side effects of bufalin. Compared to free bufalin, Bu-BCS-NPs exhibited more significant antitumor effects both by i.p. and i.v., and i.v. administration of Bu-BCS-NPs tended to be more effective than i.p. injection though no significant difference was found (Fig. 5A). More importantly, no apparent loss of body weight was found in Bu-BCS-NPs treatment (Fig. 5B). The improved antitumor effect and less toxic side effects of BuBCS-NPs were mostly contributed to EPR effect and the biotin mediated active targeting. However, it should be noted that EPR effect is observed when macromolecular drugs are administered systemically in circulation, less EPR effect will be obtained if the drugs were injected locally. Namely, i.v. injection is preferable for macromolecular drugs. In case of i.p. administration, macromolecular drugs could be partly recovered to circulation probably by macrophages and lymphatic systems, however those drugs without entering into circulation will not take the advantages of EPR effect but will retain in peritoneal cavity for a long time. This may thus result in less therapeutic effect and multiple administrations may be needed, as described in our findings showed in Fig. 5A. Moreover, besides EPR effect, the slow and sustained release property of Bu-BCS-NPs will ensure the prolonged and stable in vivo half-life of bufalin, which also partly contributed to its superior antitumor effect than free bufalin (Fig. 5). In addition, for special tumors such as peritoneal or pleural carcinomatoses, especially with ascites or pleural fluid, intracavity administration of macromolecular drugs is a useful therapeutic approach, which exhibited superior pharmacokinetic and therapeutic advantages than conventional low-molecular-weight anticancer drugs, as reported by Kimura et al. [41]. Taken together, these in vivo findings strongly suggested the potential of Bu-BCS-NPs as a new anticancer therapeutic. The advantages of Bu-BCS-NPs compared with free bufalin, e.g. water solubility, slow and sustained release of bufalin, tumor targeting and rapid intracellular uptake, will thus greatly strengthen its therapeutic applicability, not only because of its increased therapeutic effect but also because of better patient compliance (increase in administration interval or no need of repeated administration). Future studies will focus on its in vivo pharmacokinetics and in vivo anti-apoptotic actions, and its therapeutic effect will be examined in different tumor models. 4. Conclusions In this study, we successfully prepared nanoparticles of antitumor agent bufalin, Bu-BCS-NPs, by using a biotinylated chitosan. In addition to improved water solubility, the resulted nanoparticles exhibited sustained and slow release rate of bufalin, as well as superior intracellular uptake in MCF-7 cells probably attributed to the biotin modification. As the consequence, Bu-BCS-NPs exhibited much stronger cytotoxicity, as well as the effects on inducing intracellular ROS, loss of DW(m) and triggering apoptosis, in MCF-7 cells than free bufalin. In human MCF-7 breast cancer nude mice model, Bu-BCS-NPs showed markedly improved therapeutic effects, with no or less apparent side effects compared to free bufalin, benefiting from the ERP and biotin mediated tumor targeting as well as the slow release properties. Based on these findings, we thus anticipate the contribution of BCS-NPs platform to the development of bufalin toward clinical application, whereas further investigations including in vivo pharmacokinetics studies are warranted. Acknowledgements This work was supported by the Key Laboratory Supporting Program for Universities from the Department of Education of

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Please cite this article in press as: X. Tian et al., Bufalin loaded biotinylated chitosan nanoparticles: An efficient drug delivery system for targeted chemotherapy against breast carcinoma, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.05.010