Accepted Manuscript Title: Functionalized magnetic dextran-spermine nanocarriers for targeted delivery of doxorubicin to breast cancer cells Author: Shabnam Tarvirdipour Ebrahim Vasheghani-Farahani Masoud Soleimani Hassan Bardania PII: DOI: Reference:
S0378-5173(16)30106-5 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.02.012 IJP 15559
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
17-12-2015 6-2-2016 8-2-2016
Please cite this article as: Tarvirdipour, Shabnam, Vasheghani-Farahani, Ebrahim, Soleimani, Masoud, Bardania, Hassan, Functionalized magnetic dextran-spermine nanocarriers for targeted delivery of doxorubicin to breast cancer cells.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Functionalized magnetic dextran-spermine nanocarriers for targeted delivery of doxorubicin to breast cancer cells Shabnam Tarvirdipour1, Ebrahim Vasheghani-Farahani1* [email protected], Masoud Soleimani2, Hassan Bardania3 1 Biomedical Division, Faculty of Chemical Engineering, Tarbiat Modares University, P.O. Box 14115-143, Tehran, Iran 2 Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-331, Tehran,Iran 3 Cell and Molecular research Center, Yasuj University of Medical Sciences, Yasuj, Iran * Corresponding author.
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Graphical Abstract
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Abstract In recent decades, targeted drug delivery systems for breast cancer treatment emerged as an ideal alternative and promising solution to reduce systemic side effects of chemotherapeutic agents. In this study, the preparation and characterization of cationic doxorubicin (DOX) loaded magnetic dextran-spermine (DEX-SP) nanocarriers (DEX-SP-DOX) by ionic gelation were fully investigated. Then, anti-HER2 as a monoclonal antibody (mAb) and targeting ligand was conjugated via EDC/NHS reagents. The binding was confirmed by Bradford assay and further assessments were carried out by size and zeta potential measurements. Cytotoxicity effect and internalization of magnetic nanocarriers were assessed by MTT and Prussian blue assays and transmission electron microscopy (TEM), respectively. DLS measurements indicated that the size of nanocarriers increased from 62 to 84 nm by conjugation of anti-HER2 to them. The in vitro release of DOX from mAb conjugated magnetic nanocarriers at pHs 5 and 7.4 was found to be 85 and 55.5%, respectively. The MTT and Prussian blue assays demonstrated enhanced and selective uptake of DEX-SPDOX-mAb by SKBR cell (HER2 overexpressed cells) in comparison with unconjugated nanocarriers due to higher cellular binding. The TEM result also confirmed cellular internalization of DEX-SP-DOX-mAb magnetic nanocarriers. These results are very promising for targeted delivery of DOX to HER2 positive breast cancer cells.
Keywords: dextran- spermine; doxorubicin; targeted drug delivery; magnetic nanocarriers; breast
cancer.
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1. Introduction In the past few years, along with diverse development of therapeutic agents, nanotechnology as a multidisciplinary science is going through a tremendous progressive evolution in drug delivery systems (Chan, 2006; Cheng et al., 2006; Matsuda and Hunt, 2005; Williams, 2004). Accordingly, the design of novel drug delivery systems is considered to be a fundamental part of drug development with an attempt to improve the absorption, stability and therapeutic concentration of the drug within the target tissue as well as long-term controlled release of the drug at the target site (Kayser et al., 2005; Kubik et al., 2005). Hence, the nanotechnological products in the field of drug delivery result in personalized medicine as well as providing accurate diagnosis system without the limitations and disadvantages of traditional normal drugs (Sahoo et al., 2007; Vasir et al., 2005). Thus, drug loaded nanoparticles with a more selective delivery of therapeutic agents lead to superior drug efficiency with reduced drug toxicity for adjacent healthy tissue in addition to more patient comfort (de Kozak et al., 2004; Feng et al., 2004; Kattan et al., 1992). Obvious advantages of these new delivery technologies were illustrated thorough dedication of 13% of the current global pharmaceutical market to the sale of novel drug delivery products (Mazzola, 2003). Therefore, dramatic increase has occurred in application of nanomedicine with many advances during the last decades, particularly in the field of oncology due to extensive challenges to overcome systemic side effects of anticancer drugs by delivering them to their site of action while increasing therapeutic effects (Cukierman and Khan, 2010). Conventional chemotherapeutics with serious side effects due to nonspecific targeting, incapability in entering tumor’s core and lack of solubility and inability in penetrating the biological membranes result in ineffective and impaired treatment with damage of immune system and other healthy organs (Sutradhar and Amin, 2014). The mechanism of such side effects is rapid destruction of dividing cells including neoplastic cells in the digestive tract, bone 4
marrow, hair follicles and macrophages (Zhao and Rodriguez, 2013). Thus, differentiating the cancerous and normal cell via engineering the conventional chemotherapeutics in order to minimizing the side effects of chemotherapeutics agents is the major challenge of cancer therapeutics. Nanomedicine has been emerged as an area of interest to overcome the limitations of both cancer diagnosis and treatment and has made a great revolution in selective cancer targeting (Peer et al., 2007). Specific ligand-receptor targeting is one of the methods to utilize in certain cancer therapies. They can act more accurately and efficiently in probing specific tumor cells when they are attached to nanocarriers (Kukowska-Latallo et al., 2005). For example, enhanced and selective targeting to folate receptors positive cancer cells by doxorubicin-polyethylene glycol-folate conjugate micelles were successfully exhibited in vitro and in vivo (Yoo and Park, 2004). Immunoliposomes (antibody fragment-targeted liposomes) are one of the pioneer examples used for targeted delivery of doxorubicin in clinical trials (Mebiopharm Co., 2009; Sankhala et al., 2009). However, the application of this class of commonly used targeted delivery nanocarriers encountered with significant shortcoming due to the lack of allowance for sustained release of therapeutic molecules (Torchilin, 2005). Therefore new generation of nanoparticles known as theranostic systems with more patient-targeted therapies are required to overcome these problems. Moreover, nanomedicine with contrast agents especially super-paramagnetic iron oxide nanoparticles (SPIONs) (Lübbe et al., 1999) are becoming prevailing for magnetic resonance imaging (MRI) and diagnosis (Feridex®/Endorem®, Resovist®/Cliavist®) (Maeda et al., 2000), chemotherapeutics drug delivery (Doxil®, Abraxane®), gene therapy (Hosseinkhani et al., 2006), magnetic targeting, thermal therapy (Ang et al., 2009; Foy et al., 2010; Parveen et al., 2012). Previous studies have shown the stability of encapsulated SPIONs in dextran and dextran-spermine as biocompatible, hydrophilic and pH sensitive biopolymers to be used as a contrast agent (Mohammad-Taheri et al., 2012; Palmacci and Josephson, 1993; Tassa et al.,
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2011; Thorek et al., 2006) and tumor targeted drug delivery (He et al., 2013) and encapsulated BaSO4 magnetic nanoparticles in crosslinked dextran as an X-ray contrast agent for targeted delivery (Meagher et al., 2013). Surfaced modified multifunctional SPIONs vector was conjugated to several specific agents such as ligand, peptide, fluorescence and anticancer drug for different biomedical purpose (He et al., 2014). The theranostic nanosystems are the latest and pervasive generation emerging in recent years (Cole et al., 2011; Kandasamy and Maity, 2015) which use the nanocarriers with additional functionalities to achieve both imaging and therapeutic function (Xie et al., 2010). In other words, the theranostic systems would allow targeted delivery of anticancer drug and diagnosis with imaging functions using active drug delivery (Chandra et al., 2011; Huang et al., 2011; Serda et al., 2011). Different specific ligand such as mAb for overexpressed receptors on the surface of cancerous cells can be used for active targeted drug delivery. These ligands are bonded to the surface of nanocarriers via covalent conjugation (Shukla et al., 2006). In advanced breast cancer treatment, the only FDA approved system for HER2-targeted therapy is Trastuzumab (Herceptin®), a humanized mAb (Merlin et al., 2002; Montemurro et al., 2004; Pegram et al., 2004) that target HER2 receptors, overexpressed in about 30% of invasive breast cancer and not in normal tissues (Slamon et al., 1989). In addition, doxorubicin (DOX) as a chemotherapeutic medicine is extensively utilized for cancer treatment and considered one of the few drugs to be on the market in vectorized form (Doxil®/Caelyx®, PEGylated liposomal formulations) (Octavia et al., 2012). Consequently, its successful results and surpass uses make it one of the model drugs for large number of published studies in the field of nanomedicine to modify its biodistribution and clearance and reduce its cardiotoxicity which restricted its uses. Hence, an essential necessity to overcome its side effect still remains a noticeable challenge for many pharmaceutical researches (Mohan and Rapoport, 2010).
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One of the common targeting strategies is monoclonal antibodies (mAb) due to broad expression of these receptors in numerous cancer families but not in adjacent healthy tissues. The probability of blocking tumor cells signaling induces multiple researches in this domain. Thus, one of the most practical and efficient way of reducing cardiotoxic effects and enhancing the therapeutic impact of DOX is utilizing anti-HER2 functionalized nanocarriers with contrast agent in order to reach imaging and diagnosis in addition to a chemotherapeutic treatment which results in superior treatment of cancer cells via specific attachment of ligand and higher targeted DOX release (Chen et al., 2009; Xu et al., 2004). Previous studies designed targeted Herceptin-dextran iron oxide nanoparticles, human serum albumin nanoparticles (Anhorn et al., 2008; Steinhauser et al., 2006; Steinhauser et al., 2008), poly (dl-lactic acid) nanoparticles (Cirstoiu-Hapca et al., 2007), for either cancer therapy or imaging and have shown promising results. DEX-SP is a modified cationic water soluble carbohydrate biopolymer with desirable properties such as biocompatibility and biodegradability (Abedini et al., 2011) that make it as an effective polymer for multiple applications from industrial manufacturing to medicine, especially drug delivery systems and cell and tissue transfection (Hosseinkhani et al., 2004). The main reason for this high transfection capability is spermine residues (Abedini et al., 2010). This study aimed to develop doxorubicin-loaded DEX-SP magnetic nanocarriers via ionic gelation method as a theranostic system for targeted drug delivery of DOX to the breast cancer cells. The nanocarriers were conjugated by a mAb (Anti-HER2) and their cytotoxicity effect on and internalization by breast cancer cells were assessed using MTT test, Prussian blue assay and TEM analysis.
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2. Materials and methods 2.1 Materials Dextran (molecular weight 40000 Da) and 1-ethyl-3-(3-dimethyl amino-propyl) carbodiimide hydrochloride (EDC), coomassie blue, mercaptoethanol and phosphoric acid were obtained from Merk (Darmstadt, Germany), and Doxorubicin hydrochloric acid, spermine, potassium periodate, sodium borohydride, N-hydroxysuccinimide (NHS) were from Sigma-Aldrich (Germany). Anti-HER2 monoclonal antibody was purchased from Biogenex (California, USA), and Iron oxide nanoparticles (Fe3O4 aq, 8 nm) were obtained from PlasmaChem (Germany). Cell lines were provided by the Pasteur Institute, Tehran, Iran. All other chemicals were of common analytical grade.
2.2 Methods 2.2.1 DEX–SP synthesis DEX–SP conjugate was prepared by a reductive amination method as described elsewhere (Azzam et al., 2002). To prepare oxidized polysaccharide, potassium periodate was added to dextran solution at 1:1 mol ratio and stirred (6–8 h) at room temperature in the dark until a clear-yellow solution was obtained. The purification of resulting polyaldehyde derivatives was performed using dialysis membrane (cellulose tube with MW cutoff 12000) against doubly deionized water (DDW) for 2 days at -4 °C and white powder was obtained. Then the oxidized polysaccharide solution was added very slowly to a basic solution of spermine during 5–7 h. The resulting mixture was stirred gently at room temperature for 24 h. The amine-based conjugates were obtained by addition of NaBH4 to the mixture in two parts and stirring the reaction for 72h. The light-yellow mixture was dialyzed against DDW at -4 °C with 3500 MW cutoff cellulose tubing. Yellowish reduced amine-based conjugate was
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obtained from freeze drying of dialysate solution. The primary amine content was determined by the TNBS method based on a standard protocol (Snyder and Sobocinski, 1975). 2.2.2 Preparation of doxorubicin-loaded magnetic DEX-SP nanocarriers Preparation of magnetic DEX-SP-DOX nanocarriers was achieved via an ionic gelation technique (Mohammad-Taheri et al., 2012). Briefly, 2.92 mg/ml of cationic DEX-SP and DOX aqueous solution at 1:1 (w/w) ratio was mixed and titrated to the desired pH of 4.75 thorough adding 1 N HCl. Then 100 µL of Fe3O4 magnetic nanoparticles with mean diameter of 8 nm were added to DEX-SP-DOX solution and instantly sonicated for 5 min. Then, 450 μl of aqueous tripolyphosphate (TPP) solution (0.8mg/ml) by rate of 24.6 ml/min was added to the above suspension. As a result, magnetic DEX-SP-DOX nanocarriers were spontaneously formed and further mixing continued for 1 h. Finally, nanocarriers suspension was centrifuged (30000×g for 20 min) to isolate doxorubicin loaded magnetic DEX-SP nanocarriers from free drug and magnetic DEX-SP-DOX nanocarriers were washed with doubly deionized water (DDW) for 3 times in order to remove the remaining TPP. Then the anti-solvent method was used in order to increase the drug entrapment efficiency. After addition of TPP solution to the above mentioned suspension, ethanol and acetone were separately added to the synthesis medium in a volume ratio of 1:1 final suspension, and stirring continued for 1 h.
2.2.3 Characterization of the nanocarriers 2.2.3.1 Fourier transform infrared spectroscopy To study the chemical reactions between doxorubicin and DEX-SP and SPIONs entrapment, during ionic gelation, nanocarriers, DEX-SP and SPIONs were separately dried and pressed to form a tablet. The spectra was detected in KBr discs over a range of 3900-400 cm-1 and recorded by FT-IR spectrophotometer (Perkin Elmer Spectrometer Frontier, America).
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2.2.3.2 Measurement of particle size, polydispersity, and zeta potential The hydrodynamic mean diameter, polydispersity index and zeta potential of the nanocarriers were measured by dynamic light scattering (DLS) at a wavelength of 633 nm at 25°C with an angle detection of 90° using a Malvern Zetasizer (Nano-ZS, Malvern, UK). In order to provide a homogenous suspension of nanocarriers for measurement, the DOX-loaded magnetic DEX-SP nanocarriers were suspended in 1 ml DDW and sonicated for 1 minute. All measurements were done in triplicate.
2.2.3.3 Field emission scanning electron microscopy The morphology of nanocarriers was determined by a FE-SEM (S-4160, Hitachi, Japan). For this purpose, 200 µl of the suspended DOX-loaded magnetic DEX-SP nanocarriers in 2 ml purified water were sonicated for 1min. A drop of prepared suspension was layered on the FE-SEM stub and allowed to dry in air at room temperature, then dried nanocarriers were coated with the gold metal.
2.2.4 Determination of entrapment and loading efficiency After ultra-centrifugation of drug loaded nanocarriers at 30000×g, the absorbance of supernatant was measured by a UV–visible spectrophotometer (Varian Cary 50 - UV/VisSpectrometer, England) at a wavelength of 480 nm. The entrapment efficiency (EE) and drug loading efficiency (LE) were calculated by the following equations: ( )
(
( )
(
–
)
(
) – (
) )
2.2.5 Functionalizing of doxorubicin-loaded magnetic DEX-SP nanocarriers
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(1) (2)
Functionalization of doxorubicin loaded nanocarriers by anti-HER2 was carried out by EDCNHS chemistry according to Grabarek and Gergely method (Hermanson, 2013). Briefly, 500 µL of anti-HER2 solution (dissolved in NaCl-MES buffer (pH 5.8)) was added to 2 M EDC and 5 M NHS solution and stirred for 15 minutes. Then doxorubicin loaded nanocarriers, suspended in NaCl-MES buffer, were added to EDC/NHS solution and incubated at room temperature (25°C) for 2.5 h. Finally, Functionalized nanocarriers were centrifuged for 10 min at 17000×g to remove excess EDC and unconjugated anti-HER2. The binding of antiHER2 was confirmed by measuring average particle size and zeta potential using DLS method. Conjugated nanocarriers were suspended in 1ml DDW and sonicated for 1 minute to measure the size and zeta potential. The efficiency of anti-HER2 conjugation was determined by Bradford assay as described in Kruger method (Kruger, 2009).
2.2.6 In vitro drug release study In vitro doxorubicin release behavior was studied using dialysis method in phosphate buffered saline (PBS, pH 7.4) and acetate buffer (pH 5). Briefly, 1 ml of doxorubicin loaded nanocarriers was sealed in a dialysis bag (MWCO 12000 Da) and dialyzed against 15 ml of the release medium at 37 ºC in a shaking water bath. At predetermined interval times, 2 ml of medium was removed and replaced with the same amount of fresh medium. The amount of doxorubicin release was determined with a UV spectrophotometer at a wavelength of 480 nm.
2.2.7 In vitro cytotoxicity assay The cytotoxicity experiments against HER2 positive human breast cancer cell line (SKBR3) were evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded to a 96-well plate at a density of 2500 cell/100 µl/well and
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incubated for 24 h. Different concentrations of DEX-SP-DOX, DEX-SP-mAb, DEX-SPDOX-mAb samples (5, 25, 50 µg/ml) and free doxorubicin (0.1-1µg/ml) were added to cultured cells. Untreated Cells grown in the medium were used as reference value with 100 % viability. The cytotoxicity effect of nanocarriers was evaluated after 24, 48 and 72 hours of exposure. The culture medium was replaced with MTT solution (0.5mg/ml) and incubated at 37 °C for 4 h to form purple formazan crystals. Then, 150 µl of DMSO was added to wells and the absorbance of each well at a test wavelength of 570 nm was measured by ELIZA reader. The viability of treated and control cells was calculated using Eq. (3). All experiments were carried out in triplicate.
( )
(
(
)
)
(3)
The specificity of the mAb conjugated nanocarriers was investigated by comparing the cellular uptake of DEX-SP-DOX, DEX-SP-mAb, DEX-SP-DOX-mAb, and free DOX by HER2-overexpressing SKBR3 cells.
2.2.8 Prussian Blue Staining assay of Cells in Culture Iron visualization, Prussian Blue Staining assay based on the differential uptake of ferric iron by cells was assessed to demonstrate ferric iron in tissue sections. As described earlier, SKBR3 cells were cultured at a density of 5000 cells/100 µl/well in 96well culture plates. After 4 hours, cells were treated with antibody conjugated and non-conjugated nanocarriers (30µg/ml). After 14 h, cell culture medium was carefully removed and 100 ml of formalin 10 % (v/v) was added to each well to fix cells. The fixed cells were removed from formalin and 200 μL of Prussian blue solution (containing 5% Potassium ferrocyanide and 5%
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hydrochloric acid) was added to each well. After a few minutes, blue stain was appeared and imaging was performed by optical microscopy.
2.2.9 Study of cellular internalization of nanocarriers A transmission electron microscope (TEM, Philips, CM30, 300kV) was used to confirm the internalization of nanocarriers into SKBR3 cells through specific cellular uptake. Briefly, monolayer grown SKBR3 cells at a density of 8×105 in T-75 flask containing DMEM supplemented with 10 % (v/v) FBS, were treated with 1 mg/ml nanocarriers for 24 h. The cells were completely washed with PBS buffer three times after discarding the medium containing magnetic nanocarriers that were not taken up. Then collected SKBR3 cells were centrifuged at 1200g for 5 min and the supernatant was removed. After that, primary fixation was done by 2.5 % (v/v) gluteraldehyde and 4 % (v/v) formaldehyde in PBS buffer for 3 h in the dark under room temperature. The cells were then rinsed with 0.1 M PBS and embedded in 4 % osmium tetroxide solution and 2 % agarose gel for 1 h. Dehydration in a gradient concentration of ethanol (10, 30, 60, 70, 90, and 100 %) was done in order to remove free water. The specimen was first suspended in infiltration solution and stained with 0.5 % uranyl acetate for 1 h. Then embedding treatment was accomplished by epoxy resin. Polymerization of final embedded mixture was carried out at 60° C for 48 h. Finally, ultrathin sections (50– 70 nm) were cut by an ultramicrotome and fixed on grids. Further staining with 1% osmium tetraoxide for 3 min make the samples ready to get the images by TEM.
3. Results 3.1 Characterization of DEX–SP conjugates Conjugation of spermine to oxidized dextran was carried out using reductive amination method. Characterization of resulted polycations was performed by TNBS method as
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described earlier. The primary amine content of DEX–SP conjugate was calculated according to the calibration curve. Relatively high primary amine content of 1.24 mmol/g DEX-SP was obtained. The amine content in previous studies was reported to be 1.26 (Azzam et al., 2004) and 1.45 (Mohammad-Taheri et al., 2012) mmol/g DEX-SP.
3.2 The entrapment and loading efficiency of doxorubicin The effect of DOX/DEX-SP on the entrapment and loading efficiency of doxorubicin in the magnetic nanocarriers, calculated by Eqs (1) and (2), are given in Table 1. As expected, by increasing the amount of initial DOX in polymer solution the loading efficiency increased but the entrapment efficiency decreased. In order to increase the drug content in the polymeric network of nanocarriers, ethanol and acetone were added as anti-solvents after adding cross-linker into the synthesis medium as described earlier. The results in Fig. 1 show that both anti-solvents with negligible difference remarkably increased the entrapment efficiency at different weigh ratios of DOX to polymer solution. Thus, 1:10 weight ratio of DOX to DEX-SP in the presence of ethanol as antisolvent with highest EE% was selected for further studies. The selection of ethanol over acetone is due to its lower cytotoxicity
3.3 Characterization of nanocarriers 3.3.1 Size, zeta potential and polydispersity of DEX-SP-DOX nanocarriers The hydrodynamic mean diameter, zeta potential, and polydispersity indexes of nanocarriers, prepared in different weigh ratios of DOX to DEX-SP are given in Table 2. Based on these results, DEX-SP-DOX-1 with an acceptable size range of 50–100 nm and zeta potential and maximum EE (51%) was selected as an optimum formulation. Since particle size and surface coating have great impact on nanoparticles half-life in the blood circulation system, magnetic
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nanocarriers with proper surface coating in this specific size range (under 100 nm) are capable of escaping elimination by the body’s reticuloendothelial system (RES) (Win and Feng, 2005).
3.3.2 Fourier transforms infrared (FT-IR) spectroscopy The FT-IR spectra of pure Fe3O4, magnetic nanocarriers of DEX-SP-DOX and DEX-SP polymer are depicted in Fig. 2. The FT-IR spectrum for pure Fe3O4 shows that the absorption peaks at 580 cm-1 belong to the stretching vibration mode of Fe–O bonds (Table 3). In addition, the peak centered at 1384 cm-1 can be ascribed to C-H bending vibration mode (Duhan and Devi, 2010). The weak band centered around 1615 cm-1 is due to the bending mode of H-O-H adsorbed at the Fe3O4 surface. The intensity of absorption peaks of 580 and 631 cm-1 for pure Fe3O4 were significantly reduced after loading of these nanoparticles in polymeric nanocarriers. Furthermore, the spectrum of DEX-SP polymer shows no absorption peak in the range of 650-400 cm-1 compared with magnetic nanocarrier's absorption peak in this range. These results reflect that Fe3O4 were successfully entrapped in the polymeric network.
3.3.3 The morphology of magnetic nanocarriers The FE-SEM result for DOX-loaded magnetic nanocarriers in Fig. 3 indicates that these nanocarriers with an average size of 60±5 nm were spherical and almost well dispersed.
3.4 Conjugation of anti-HER2 mAb to DEX-SP-DOX magnetic nanocarriers To verify mAb conjugation to nanocarriers, the size and zeta potential of magnetic nanocarriers, before and after antibody conjugation, as well as the antibody were measured and corresponding results are given in Table 4. Regarding the negative zeta potential of 15
antibody, the zeta potential of nanocarriers reduced after antibody binding to nanocarriers but their size increased from 62 to 84 nm. Spectrophotometric Bradford assay was also used to investigate the binding efficiency of mAb to nanocarriers. The results of this assay confirmed 40 μg binding of antibody per 1 mg of nanocarriers with 24% efficiency. In a study on functionalized poly lactic acid nanoparticles with anti-HER2 for tumor cell targeting, 8 μg antibody/mg of nanoparticles with 16% efficiency was conjugated (Cirstoiu-Hapca et al., 2007).
3.5 In vitro release of doxorubicin from magnetic nanocarriers Fig. 4 shows DOX release profile from magnetic DEX-SP-DOX-mAb-1 nanocarriers (with the most drug entrapment efficiency). In addition, the release profile of free DOX was also investigated to evaluate slow release of nanocarriers for targeted drug delivery and prove dialysis bag non-resistancy effect (Fig. 4). The profile of DOX release from magnetic DEXSP-DOX-mAb-1 nanocarriers shows slow release pattern with a slight burst effect in the early hours. The DOX release from these magnetic nanocarriers after 6 days was about 55.5 % of total entrapped drug. In order to ensure the non-inhibitory effect of mAb (anti-HER2) binding to the surface of magnetic nanocarriers, drug release was also examined from magnetic nanocarriers without mAb antibody conjugation to their surface as well. As shown in Fig. 4, the presence of mAb antibody on the surface of magnetic nanocarriers did not impose drug release resistance and the two curves were comparatively coincided. The release test was performed in triplicate. Due to the acidic environment around the tumor, DOX release was investigated in phosphatebuffered saline (PBS, pH 7.4) and acetate buffer (pH 5) which mimics normal biologic and cancerous conditions, respectively (Fig. 5). The cancerous medium condition had a remarkable effect and DOX release from magnetic nanocarriers increased significantly as 16
expected, e.g 55.5 % drug release in normal condition compared to 85 % in cancerous condition due to positive charge of magnetic DEX-SP-DOX-mAb-1 nanocarriers and DOX with consequent repulsion at this pH value. In addition, the increase of drug release at pH 5 can be attributed to higher swelling of the network due to ionization of cationic polymer at acidic environment.
3.6 Cell viability As shown in Fig. 6, the effect of different concentration of free DOX on the viability of SKBR3 cells was evaluated and IC50 was calculated. The IC50 is the drug concentration of an inhibitor at which the cell death is reached to its half percent at the designated time. Free DOX's IC50 was about 0.338, 0.29 and 0.272 μg/ml for 24, 48 and 72 hours respectively according to the above definition. In the next step, the cytotoxicity effect of DEX-SP-DOX-, DEX-SP-mAb, DEX-SP-DOXmAb-1 and free DOX against SKBR3 cells was also investigated by MTT assay for 72h. The concentration of samples was equivalent to calculated IC50 of free DOX. The results in Fig. 7 show that DEX-SP-DOX-mAb-1 has more cytotoxicity effect on HER2-overexpressing SKBR3 cell line compared with non-targeting nanocarriers of DEX-SP-DOX and free drug. The reduced cell viability of conjugated nanocarriers confirms that efficient targeted binding of mAB to the cells is responsible for higher uptake of DEX-SP-DOX-mAb-1 magnetic nanocarriers with consequent enhanced DOX release from magnetic nanocarriers, particularly after 72 h. Moreover, lower cytotoxicity of DEX-SP-DOX and DEX-SP-mAb compared to free DOX indicated high biocompatibility of DEX-SP polymer. To verify cellular uptake of nanocarriers further, cellular studies via Prussian blue and TEM experiments were carried out.
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3.7 Detection of anti-HER2-conjugated magnetic nanocarriers uptake by breast cancer cells via Prussian blue assay Additional assessment for interaction of anti-HER2-conjugated nanocarriers with HER2 over expressed breast cancer cells (SKBR3) and was carried out via Prussian blue iron staining assay to confirm higher uptake of conjugated nanocarriers by cells as a result of anti-HER2 mAb (a targeting ligand) conjugation. SKBR3 cells were imaged under light microscopy with magnification of 40 after treatment with anti-HER2-conjugated and non-conjugated magnetic nanocarriers and stained with Prussian blue solution as described earlier to demonstrate ferric iron uptake of cells. Cells marked by magnetic DEX-SP-DOX-mAb-1 nanocarriers and magnetic DEX-SP-DOX-1 nanocarriers are shown with blue points in Fig. 8. The results indicate that the higher percentages of cells were marked with the anti-HER2-conjugated magnetic nanocarriers compared with non-conjugated magnetic nanocarriers. Furthermore, translocation and cellular uptake of the targeted nanocarriers differ from those of free antibody ones. According to the images shown in Fig. 8, targeting magnetic nanocarriers demonstrated a greater qualitative amount of translocation and cellular uptake.
3.8 Transmission electron microscopy study TEM images in Fig. 9 were taken to show internalization of targeting nanocarriers into SKBR3 cells through specific cellular uptake. Arrows are used to show the localization of some magnetic nanocarriers within the cells as well as the accumulation of nanocarriers, observed in different regions of the cell.
4. Discussion In recent decades, the use of DOX as a chemotherapeutic drug with wide spectrum of anticancer activities for targeted drug delivery systems based on MNPs has been aroused 18
widespread and reported so well by extended knowledge of its chemical properties (Lu et al., 2012; Sanson et al., 2010; Yang et al., 2011). This study aimed to develop a theranostic system using a cationic polysaccharide, DEX-SP with various desirable properties. The primary amine content of DEX–SP conjugate by TNBS method represented an acceptable value of 1.24 mmol amine/g polymer (Azzam et al., 2002). The synthesis of DOX-loaded DEX-SP magnetic nanocarriers were carried out via ionic gelation method in the presence of TPP as a crosslinker. Such self- assembly method for efficient entrapment of super paramagnetic iron oxide and DOX
has been previously
reported in respectively
poly(ethylene glycol)-block-poly(ɛ-caprolactone) (PEG-PCL) nanomicelles, poly (ethylene oxide)-trimellitic anhydride chloride-folate (PEO-TMA-FA), hydrophobically modified glycol chitosans nanoparticles (Liao et al., 2011; Maeng et al., 2010; Park et al., 2006). Different weigh ratios of DOX to DEX-SP, with or without anti-solvent in the synthesis medium, were used for drug entrapment in nanocarriers. The loading efficiency decreased by increasing DOX to polymer ratio due to limited solubility of drug in polymer solution. The entrapment of DOX in nanocarriers increased in the presence of anti-solvents as a result of solvent removal from polymeric solution in which the drug was dissolved. The zeta potential of the nanocarriers did not change considerably by variation of DOX/polymer ratio, but the hydrodynamic diameter of the nanocarriers increased by increase of DOX to DEX-SP ratio. The polydispersity index was adequately low (
S0378-5173(16)30106-5 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.02.012 IJP 15559
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
17-12-2015 6-2-2016 8-2-2016
Please cite this article as: Tarvirdipour, Shabnam, Vasheghani-Farahani, Ebrahim, Soleimani, Masoud, Bardania, Hassan, Functionalized magnetic dextran-spermine nanocarriers for targeted delivery of doxorubicin to breast cancer cells.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Functionalized magnetic dextran-spermine nanocarriers for targeted delivery of doxorubicin to breast cancer cells Shabnam Tarvirdipour1, Ebrahim Vasheghani-Farahani1* [email protected], Masoud Soleimani2, Hassan Bardania3 1 Biomedical Division, Faculty of Chemical Engineering, Tarbiat Modares University, P.O. Box 14115-143, Tehran, Iran 2 Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-331, Tehran,Iran 3 Cell and Molecular research Center, Yasuj University of Medical Sciences, Yasuj, Iran * Corresponding author.
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Graphical Abstract
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Abstract In recent decades, targeted drug delivery systems for breast cancer treatment emerged as an ideal alternative and promising solution to reduce systemic side effects of chemotherapeutic agents. In this study, the preparation and characterization of cationic doxorubicin (DOX) loaded magnetic dextran-spermine (DEX-SP) nanocarriers (DEX-SP-DOX) by ionic gelation were fully investigated. Then, anti-HER2 as a monoclonal antibody (mAb) and targeting ligand was conjugated via EDC/NHS reagents. The binding was confirmed by Bradford assay and further assessments were carried out by size and zeta potential measurements. Cytotoxicity effect and internalization of magnetic nanocarriers were assessed by MTT and Prussian blue assays and transmission electron microscopy (TEM), respectively. DLS measurements indicated that the size of nanocarriers increased from 62 to 84 nm by conjugation of anti-HER2 to them. The in vitro release of DOX from mAb conjugated magnetic nanocarriers at pHs 5 and 7.4 was found to be 85 and 55.5%, respectively. The MTT and Prussian blue assays demonstrated enhanced and selective uptake of DEX-SPDOX-mAb by SKBR cell (HER2 overexpressed cells) in comparison with unconjugated nanocarriers due to higher cellular binding. The TEM result also confirmed cellular internalization of DEX-SP-DOX-mAb magnetic nanocarriers. These results are very promising for targeted delivery of DOX to HER2 positive breast cancer cells.
Keywords: dextran- spermine; doxorubicin; targeted drug delivery; magnetic nanocarriers; breast
cancer.
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1. Introduction In the past few years, along with diverse development of therapeutic agents, nanotechnology as a multidisciplinary science is going through a tremendous progressive evolution in drug delivery systems (Chan, 2006; Cheng et al., 2006; Matsuda and Hunt, 2005; Williams, 2004). Accordingly, the design of novel drug delivery systems is considered to be a fundamental part of drug development with an attempt to improve the absorption, stability and therapeutic concentration of the drug within the target tissue as well as long-term controlled release of the drug at the target site (Kayser et al., 2005; Kubik et al., 2005). Hence, the nanotechnological products in the field of drug delivery result in personalized medicine as well as providing accurate diagnosis system without the limitations and disadvantages of traditional normal drugs (Sahoo et al., 2007; Vasir et al., 2005). Thus, drug loaded nanoparticles with a more selective delivery of therapeutic agents lead to superior drug efficiency with reduced drug toxicity for adjacent healthy tissue in addition to more patient comfort (de Kozak et al., 2004; Feng et al., 2004; Kattan et al., 1992). Obvious advantages of these new delivery technologies were illustrated thorough dedication of 13% of the current global pharmaceutical market to the sale of novel drug delivery products (Mazzola, 2003). Therefore, dramatic increase has occurred in application of nanomedicine with many advances during the last decades, particularly in the field of oncology due to extensive challenges to overcome systemic side effects of anticancer drugs by delivering them to their site of action while increasing therapeutic effects (Cukierman and Khan, 2010). Conventional chemotherapeutics with serious side effects due to nonspecific targeting, incapability in entering tumor’s core and lack of solubility and inability in penetrating the biological membranes result in ineffective and impaired treatment with damage of immune system and other healthy organs (Sutradhar and Amin, 2014). The mechanism of such side effects is rapid destruction of dividing cells including neoplastic cells in the digestive tract, bone 4
marrow, hair follicles and macrophages (Zhao and Rodriguez, 2013). Thus, differentiating the cancerous and normal cell via engineering the conventional chemotherapeutics in order to minimizing the side effects of chemotherapeutics agents is the major challenge of cancer therapeutics. Nanomedicine has been emerged as an area of interest to overcome the limitations of both cancer diagnosis and treatment and has made a great revolution in selective cancer targeting (Peer et al., 2007). Specific ligand-receptor targeting is one of the methods to utilize in certain cancer therapies. They can act more accurately and efficiently in probing specific tumor cells when they are attached to nanocarriers (Kukowska-Latallo et al., 2005). For example, enhanced and selective targeting to folate receptors positive cancer cells by doxorubicin-polyethylene glycol-folate conjugate micelles were successfully exhibited in vitro and in vivo (Yoo and Park, 2004). Immunoliposomes (antibody fragment-targeted liposomes) are one of the pioneer examples used for targeted delivery of doxorubicin in clinical trials (Mebiopharm Co., 2009; Sankhala et al., 2009). However, the application of this class of commonly used targeted delivery nanocarriers encountered with significant shortcoming due to the lack of allowance for sustained release of therapeutic molecules (Torchilin, 2005). Therefore new generation of nanoparticles known as theranostic systems with more patient-targeted therapies are required to overcome these problems. Moreover, nanomedicine with contrast agents especially super-paramagnetic iron oxide nanoparticles (SPIONs) (Lübbe et al., 1999) are becoming prevailing for magnetic resonance imaging (MRI) and diagnosis (Feridex®/Endorem®, Resovist®/Cliavist®) (Maeda et al., 2000), chemotherapeutics drug delivery (Doxil®, Abraxane®), gene therapy (Hosseinkhani et al., 2006), magnetic targeting, thermal therapy (Ang et al., 2009; Foy et al., 2010; Parveen et al., 2012). Previous studies have shown the stability of encapsulated SPIONs in dextran and dextran-spermine as biocompatible, hydrophilic and pH sensitive biopolymers to be used as a contrast agent (Mohammad-Taheri et al., 2012; Palmacci and Josephson, 1993; Tassa et al.,
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2011; Thorek et al., 2006) and tumor targeted drug delivery (He et al., 2013) and encapsulated BaSO4 magnetic nanoparticles in crosslinked dextran as an X-ray contrast agent for targeted delivery (Meagher et al., 2013). Surfaced modified multifunctional SPIONs vector was conjugated to several specific agents such as ligand, peptide, fluorescence and anticancer drug for different biomedical purpose (He et al., 2014). The theranostic nanosystems are the latest and pervasive generation emerging in recent years (Cole et al., 2011; Kandasamy and Maity, 2015) which use the nanocarriers with additional functionalities to achieve both imaging and therapeutic function (Xie et al., 2010). In other words, the theranostic systems would allow targeted delivery of anticancer drug and diagnosis with imaging functions using active drug delivery (Chandra et al., 2011; Huang et al., 2011; Serda et al., 2011). Different specific ligand such as mAb for overexpressed receptors on the surface of cancerous cells can be used for active targeted drug delivery. These ligands are bonded to the surface of nanocarriers via covalent conjugation (Shukla et al., 2006). In advanced breast cancer treatment, the only FDA approved system for HER2-targeted therapy is Trastuzumab (Herceptin®), a humanized mAb (Merlin et al., 2002; Montemurro et al., 2004; Pegram et al., 2004) that target HER2 receptors, overexpressed in about 30% of invasive breast cancer and not in normal tissues (Slamon et al., 1989). In addition, doxorubicin (DOX) as a chemotherapeutic medicine is extensively utilized for cancer treatment and considered one of the few drugs to be on the market in vectorized form (Doxil®/Caelyx®, PEGylated liposomal formulations) (Octavia et al., 2012). Consequently, its successful results and surpass uses make it one of the model drugs for large number of published studies in the field of nanomedicine to modify its biodistribution and clearance and reduce its cardiotoxicity which restricted its uses. Hence, an essential necessity to overcome its side effect still remains a noticeable challenge for many pharmaceutical researches (Mohan and Rapoport, 2010).
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One of the common targeting strategies is monoclonal antibodies (mAb) due to broad expression of these receptors in numerous cancer families but not in adjacent healthy tissues. The probability of blocking tumor cells signaling induces multiple researches in this domain. Thus, one of the most practical and efficient way of reducing cardiotoxic effects and enhancing the therapeutic impact of DOX is utilizing anti-HER2 functionalized nanocarriers with contrast agent in order to reach imaging and diagnosis in addition to a chemotherapeutic treatment which results in superior treatment of cancer cells via specific attachment of ligand and higher targeted DOX release (Chen et al., 2009; Xu et al., 2004). Previous studies designed targeted Herceptin-dextran iron oxide nanoparticles, human serum albumin nanoparticles (Anhorn et al., 2008; Steinhauser et al., 2006; Steinhauser et al., 2008), poly (dl-lactic acid) nanoparticles (Cirstoiu-Hapca et al., 2007), for either cancer therapy or imaging and have shown promising results. DEX-SP is a modified cationic water soluble carbohydrate biopolymer with desirable properties such as biocompatibility and biodegradability (Abedini et al., 2011) that make it as an effective polymer for multiple applications from industrial manufacturing to medicine, especially drug delivery systems and cell and tissue transfection (Hosseinkhani et al., 2004). The main reason for this high transfection capability is spermine residues (Abedini et al., 2010). This study aimed to develop doxorubicin-loaded DEX-SP magnetic nanocarriers via ionic gelation method as a theranostic system for targeted drug delivery of DOX to the breast cancer cells. The nanocarriers were conjugated by a mAb (Anti-HER2) and their cytotoxicity effect on and internalization by breast cancer cells were assessed using MTT test, Prussian blue assay and TEM analysis.
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2. Materials and methods 2.1 Materials Dextran (molecular weight 40000 Da) and 1-ethyl-3-(3-dimethyl amino-propyl) carbodiimide hydrochloride (EDC), coomassie blue, mercaptoethanol and phosphoric acid were obtained from Merk (Darmstadt, Germany), and Doxorubicin hydrochloric acid, spermine, potassium periodate, sodium borohydride, N-hydroxysuccinimide (NHS) were from Sigma-Aldrich (Germany). Anti-HER2 monoclonal antibody was purchased from Biogenex (California, USA), and Iron oxide nanoparticles (Fe3O4 aq, 8 nm) were obtained from PlasmaChem (Germany). Cell lines were provided by the Pasteur Institute, Tehran, Iran. All other chemicals were of common analytical grade.
2.2 Methods 2.2.1 DEX–SP synthesis DEX–SP conjugate was prepared by a reductive amination method as described elsewhere (Azzam et al., 2002). To prepare oxidized polysaccharide, potassium periodate was added to dextran solution at 1:1 mol ratio and stirred (6–8 h) at room temperature in the dark until a clear-yellow solution was obtained. The purification of resulting polyaldehyde derivatives was performed using dialysis membrane (cellulose tube with MW cutoff 12000) against doubly deionized water (DDW) for 2 days at -4 °C and white powder was obtained. Then the oxidized polysaccharide solution was added very slowly to a basic solution of spermine during 5–7 h. The resulting mixture was stirred gently at room temperature for 24 h. The amine-based conjugates were obtained by addition of NaBH4 to the mixture in two parts and stirring the reaction for 72h. The light-yellow mixture was dialyzed against DDW at -4 °C with 3500 MW cutoff cellulose tubing. Yellowish reduced amine-based conjugate was
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obtained from freeze drying of dialysate solution. The primary amine content was determined by the TNBS method based on a standard protocol (Snyder and Sobocinski, 1975). 2.2.2 Preparation of doxorubicin-loaded magnetic DEX-SP nanocarriers Preparation of magnetic DEX-SP-DOX nanocarriers was achieved via an ionic gelation technique (Mohammad-Taheri et al., 2012). Briefly, 2.92 mg/ml of cationic DEX-SP and DOX aqueous solution at 1:1 (w/w) ratio was mixed and titrated to the desired pH of 4.75 thorough adding 1 N HCl. Then 100 µL of Fe3O4 magnetic nanoparticles with mean diameter of 8 nm were added to DEX-SP-DOX solution and instantly sonicated for 5 min. Then, 450 μl of aqueous tripolyphosphate (TPP) solution (0.8mg/ml) by rate of 24.6 ml/min was added to the above suspension. As a result, magnetic DEX-SP-DOX nanocarriers were spontaneously formed and further mixing continued for 1 h. Finally, nanocarriers suspension was centrifuged (30000×g for 20 min) to isolate doxorubicin loaded magnetic DEX-SP nanocarriers from free drug and magnetic DEX-SP-DOX nanocarriers were washed with doubly deionized water (DDW) for 3 times in order to remove the remaining TPP. Then the anti-solvent method was used in order to increase the drug entrapment efficiency. After addition of TPP solution to the above mentioned suspension, ethanol and acetone were separately added to the synthesis medium in a volume ratio of 1:1 final suspension, and stirring continued for 1 h.
2.2.3 Characterization of the nanocarriers 2.2.3.1 Fourier transform infrared spectroscopy To study the chemical reactions between doxorubicin and DEX-SP and SPIONs entrapment, during ionic gelation, nanocarriers, DEX-SP and SPIONs were separately dried and pressed to form a tablet. The spectra was detected in KBr discs over a range of 3900-400 cm-1 and recorded by FT-IR spectrophotometer (Perkin Elmer Spectrometer Frontier, America).
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2.2.3.2 Measurement of particle size, polydispersity, and zeta potential The hydrodynamic mean diameter, polydispersity index and zeta potential of the nanocarriers were measured by dynamic light scattering (DLS) at a wavelength of 633 nm at 25°C with an angle detection of 90° using a Malvern Zetasizer (Nano-ZS, Malvern, UK). In order to provide a homogenous suspension of nanocarriers for measurement, the DOX-loaded magnetic DEX-SP nanocarriers were suspended in 1 ml DDW and sonicated for 1 minute. All measurements were done in triplicate.
2.2.3.3 Field emission scanning electron microscopy The morphology of nanocarriers was determined by a FE-SEM (S-4160, Hitachi, Japan). For this purpose, 200 µl of the suspended DOX-loaded magnetic DEX-SP nanocarriers in 2 ml purified water were sonicated for 1min. A drop of prepared suspension was layered on the FE-SEM stub and allowed to dry in air at room temperature, then dried nanocarriers were coated with the gold metal.
2.2.4 Determination of entrapment and loading efficiency After ultra-centrifugation of drug loaded nanocarriers at 30000×g, the absorbance of supernatant was measured by a UV–visible spectrophotometer (Varian Cary 50 - UV/VisSpectrometer, England) at a wavelength of 480 nm. The entrapment efficiency (EE) and drug loading efficiency (LE) were calculated by the following equations: ( )
(
( )
(
–
)
(
) – (
) )
2.2.5 Functionalizing of doxorubicin-loaded magnetic DEX-SP nanocarriers
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(1) (2)
Functionalization of doxorubicin loaded nanocarriers by anti-HER2 was carried out by EDCNHS chemistry according to Grabarek and Gergely method (Hermanson, 2013). Briefly, 500 µL of anti-HER2 solution (dissolved in NaCl-MES buffer (pH 5.8)) was added to 2 M EDC and 5 M NHS solution and stirred for 15 minutes. Then doxorubicin loaded nanocarriers, suspended in NaCl-MES buffer, were added to EDC/NHS solution and incubated at room temperature (25°C) for 2.5 h. Finally, Functionalized nanocarriers were centrifuged for 10 min at 17000×g to remove excess EDC and unconjugated anti-HER2. The binding of antiHER2 was confirmed by measuring average particle size and zeta potential using DLS method. Conjugated nanocarriers were suspended in 1ml DDW and sonicated for 1 minute to measure the size and zeta potential. The efficiency of anti-HER2 conjugation was determined by Bradford assay as described in Kruger method (Kruger, 2009).
2.2.6 In vitro drug release study In vitro doxorubicin release behavior was studied using dialysis method in phosphate buffered saline (PBS, pH 7.4) and acetate buffer (pH 5). Briefly, 1 ml of doxorubicin loaded nanocarriers was sealed in a dialysis bag (MWCO 12000 Da) and dialyzed against 15 ml of the release medium at 37 ºC in a shaking water bath. At predetermined interval times, 2 ml of medium was removed and replaced with the same amount of fresh medium. The amount of doxorubicin release was determined with a UV spectrophotometer at a wavelength of 480 nm.
2.2.7 In vitro cytotoxicity assay The cytotoxicity experiments against HER2 positive human breast cancer cell line (SKBR3) were evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded to a 96-well plate at a density of 2500 cell/100 µl/well and
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incubated for 24 h. Different concentrations of DEX-SP-DOX, DEX-SP-mAb, DEX-SPDOX-mAb samples (5, 25, 50 µg/ml) and free doxorubicin (0.1-1µg/ml) were added to cultured cells. Untreated Cells grown in the medium were used as reference value with 100 % viability. The cytotoxicity effect of nanocarriers was evaluated after 24, 48 and 72 hours of exposure. The culture medium was replaced with MTT solution (0.5mg/ml) and incubated at 37 °C for 4 h to form purple formazan crystals. Then, 150 µl of DMSO was added to wells and the absorbance of each well at a test wavelength of 570 nm was measured by ELIZA reader. The viability of treated and control cells was calculated using Eq. (3). All experiments were carried out in triplicate.
( )
(
(
)
)
(3)
The specificity of the mAb conjugated nanocarriers was investigated by comparing the cellular uptake of DEX-SP-DOX, DEX-SP-mAb, DEX-SP-DOX-mAb, and free DOX by HER2-overexpressing SKBR3 cells.
2.2.8 Prussian Blue Staining assay of Cells in Culture Iron visualization, Prussian Blue Staining assay based on the differential uptake of ferric iron by cells was assessed to demonstrate ferric iron in tissue sections. As described earlier, SKBR3 cells were cultured at a density of 5000 cells/100 µl/well in 96well culture plates. After 4 hours, cells were treated with antibody conjugated and non-conjugated nanocarriers (30µg/ml). After 14 h, cell culture medium was carefully removed and 100 ml of formalin 10 % (v/v) was added to each well to fix cells. The fixed cells were removed from formalin and 200 μL of Prussian blue solution (containing 5% Potassium ferrocyanide and 5%
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hydrochloric acid) was added to each well. After a few minutes, blue stain was appeared and imaging was performed by optical microscopy.
2.2.9 Study of cellular internalization of nanocarriers A transmission electron microscope (TEM, Philips, CM30, 300kV) was used to confirm the internalization of nanocarriers into SKBR3 cells through specific cellular uptake. Briefly, monolayer grown SKBR3 cells at a density of 8×105 in T-75 flask containing DMEM supplemented with 10 % (v/v) FBS, were treated with 1 mg/ml nanocarriers for 24 h. The cells were completely washed with PBS buffer three times after discarding the medium containing magnetic nanocarriers that were not taken up. Then collected SKBR3 cells were centrifuged at 1200g for 5 min and the supernatant was removed. After that, primary fixation was done by 2.5 % (v/v) gluteraldehyde and 4 % (v/v) formaldehyde in PBS buffer for 3 h in the dark under room temperature. The cells were then rinsed with 0.1 M PBS and embedded in 4 % osmium tetroxide solution and 2 % agarose gel for 1 h. Dehydration in a gradient concentration of ethanol (10, 30, 60, 70, 90, and 100 %) was done in order to remove free water. The specimen was first suspended in infiltration solution and stained with 0.5 % uranyl acetate for 1 h. Then embedding treatment was accomplished by epoxy resin. Polymerization of final embedded mixture was carried out at 60° C for 48 h. Finally, ultrathin sections (50– 70 nm) were cut by an ultramicrotome and fixed on grids. Further staining with 1% osmium tetraoxide for 3 min make the samples ready to get the images by TEM.
3. Results 3.1 Characterization of DEX–SP conjugates Conjugation of spermine to oxidized dextran was carried out using reductive amination method. Characterization of resulted polycations was performed by TNBS method as
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described earlier. The primary amine content of DEX–SP conjugate was calculated according to the calibration curve. Relatively high primary amine content of 1.24 mmol/g DEX-SP was obtained. The amine content in previous studies was reported to be 1.26 (Azzam et al., 2004) and 1.45 (Mohammad-Taheri et al., 2012) mmol/g DEX-SP.
3.2 The entrapment and loading efficiency of doxorubicin The effect of DOX/DEX-SP on the entrapment and loading efficiency of doxorubicin in the magnetic nanocarriers, calculated by Eqs (1) and (2), are given in Table 1. As expected, by increasing the amount of initial DOX in polymer solution the loading efficiency increased but the entrapment efficiency decreased. In order to increase the drug content in the polymeric network of nanocarriers, ethanol and acetone were added as anti-solvents after adding cross-linker into the synthesis medium as described earlier. The results in Fig. 1 show that both anti-solvents with negligible difference remarkably increased the entrapment efficiency at different weigh ratios of DOX to polymer solution. Thus, 1:10 weight ratio of DOX to DEX-SP in the presence of ethanol as antisolvent with highest EE% was selected for further studies. The selection of ethanol over acetone is due to its lower cytotoxicity
3.3 Characterization of nanocarriers 3.3.1 Size, zeta potential and polydispersity of DEX-SP-DOX nanocarriers The hydrodynamic mean diameter, zeta potential, and polydispersity indexes of nanocarriers, prepared in different weigh ratios of DOX to DEX-SP are given in Table 2. Based on these results, DEX-SP-DOX-1 with an acceptable size range of 50–100 nm and zeta potential and maximum EE (51%) was selected as an optimum formulation. Since particle size and surface coating have great impact on nanoparticles half-life in the blood circulation system, magnetic
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nanocarriers with proper surface coating in this specific size range (under 100 nm) are capable of escaping elimination by the body’s reticuloendothelial system (RES) (Win and Feng, 2005).
3.3.2 Fourier transforms infrared (FT-IR) spectroscopy The FT-IR spectra of pure Fe3O4, magnetic nanocarriers of DEX-SP-DOX and DEX-SP polymer are depicted in Fig. 2. The FT-IR spectrum for pure Fe3O4 shows that the absorption peaks at 580 cm-1 belong to the stretching vibration mode of Fe–O bonds (Table 3). In addition, the peak centered at 1384 cm-1 can be ascribed to C-H bending vibration mode (Duhan and Devi, 2010). The weak band centered around 1615 cm-1 is due to the bending mode of H-O-H adsorbed at the Fe3O4 surface. The intensity of absorption peaks of 580 and 631 cm-1 for pure Fe3O4 were significantly reduced after loading of these nanoparticles in polymeric nanocarriers. Furthermore, the spectrum of DEX-SP polymer shows no absorption peak in the range of 650-400 cm-1 compared with magnetic nanocarrier's absorption peak in this range. These results reflect that Fe3O4 were successfully entrapped in the polymeric network.
3.3.3 The morphology of magnetic nanocarriers The FE-SEM result for DOX-loaded magnetic nanocarriers in Fig. 3 indicates that these nanocarriers with an average size of 60±5 nm were spherical and almost well dispersed.
3.4 Conjugation of anti-HER2 mAb to DEX-SP-DOX magnetic nanocarriers To verify mAb conjugation to nanocarriers, the size and zeta potential of magnetic nanocarriers, before and after antibody conjugation, as well as the antibody were measured and corresponding results are given in Table 4. Regarding the negative zeta potential of 15
antibody, the zeta potential of nanocarriers reduced after antibody binding to nanocarriers but their size increased from 62 to 84 nm. Spectrophotometric Bradford assay was also used to investigate the binding efficiency of mAb to nanocarriers. The results of this assay confirmed 40 μg binding of antibody per 1 mg of nanocarriers with 24% efficiency. In a study on functionalized poly lactic acid nanoparticles with anti-HER2 for tumor cell targeting, 8 μg antibody/mg of nanoparticles with 16% efficiency was conjugated (Cirstoiu-Hapca et al., 2007).
3.5 In vitro release of doxorubicin from magnetic nanocarriers Fig. 4 shows DOX release profile from magnetic DEX-SP-DOX-mAb-1 nanocarriers (with the most drug entrapment efficiency). In addition, the release profile of free DOX was also investigated to evaluate slow release of nanocarriers for targeted drug delivery and prove dialysis bag non-resistancy effect (Fig. 4). The profile of DOX release from magnetic DEXSP-DOX-mAb-1 nanocarriers shows slow release pattern with a slight burst effect in the early hours. The DOX release from these magnetic nanocarriers after 6 days was about 55.5 % of total entrapped drug. In order to ensure the non-inhibitory effect of mAb (anti-HER2) binding to the surface of magnetic nanocarriers, drug release was also examined from magnetic nanocarriers without mAb antibody conjugation to their surface as well. As shown in Fig. 4, the presence of mAb antibody on the surface of magnetic nanocarriers did not impose drug release resistance and the two curves were comparatively coincided. The release test was performed in triplicate. Due to the acidic environment around the tumor, DOX release was investigated in phosphatebuffered saline (PBS, pH 7.4) and acetate buffer (pH 5) which mimics normal biologic and cancerous conditions, respectively (Fig. 5). The cancerous medium condition had a remarkable effect and DOX release from magnetic nanocarriers increased significantly as 16
expected, e.g 55.5 % drug release in normal condition compared to 85 % in cancerous condition due to positive charge of magnetic DEX-SP-DOX-mAb-1 nanocarriers and DOX with consequent repulsion at this pH value. In addition, the increase of drug release at pH 5 can be attributed to higher swelling of the network due to ionization of cationic polymer at acidic environment.
3.6 Cell viability As shown in Fig. 6, the effect of different concentration of free DOX on the viability of SKBR3 cells was evaluated and IC50 was calculated. The IC50 is the drug concentration of an inhibitor at which the cell death is reached to its half percent at the designated time. Free DOX's IC50 was about 0.338, 0.29 and 0.272 μg/ml for 24, 48 and 72 hours respectively according to the above definition. In the next step, the cytotoxicity effect of DEX-SP-DOX-, DEX-SP-mAb, DEX-SP-DOXmAb-1 and free DOX against SKBR3 cells was also investigated by MTT assay for 72h. The concentration of samples was equivalent to calculated IC50 of free DOX. The results in Fig. 7 show that DEX-SP-DOX-mAb-1 has more cytotoxicity effect on HER2-overexpressing SKBR3 cell line compared with non-targeting nanocarriers of DEX-SP-DOX and free drug. The reduced cell viability of conjugated nanocarriers confirms that efficient targeted binding of mAB to the cells is responsible for higher uptake of DEX-SP-DOX-mAb-1 magnetic nanocarriers with consequent enhanced DOX release from magnetic nanocarriers, particularly after 72 h. Moreover, lower cytotoxicity of DEX-SP-DOX and DEX-SP-mAb compared to free DOX indicated high biocompatibility of DEX-SP polymer. To verify cellular uptake of nanocarriers further, cellular studies via Prussian blue and TEM experiments were carried out.
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3.7 Detection of anti-HER2-conjugated magnetic nanocarriers uptake by breast cancer cells via Prussian blue assay Additional assessment for interaction of anti-HER2-conjugated nanocarriers with HER2 over expressed breast cancer cells (SKBR3) and was carried out via Prussian blue iron staining assay to confirm higher uptake of conjugated nanocarriers by cells as a result of anti-HER2 mAb (a targeting ligand) conjugation. SKBR3 cells were imaged under light microscopy with magnification of 40 after treatment with anti-HER2-conjugated and non-conjugated magnetic nanocarriers and stained with Prussian blue solution as described earlier to demonstrate ferric iron uptake of cells. Cells marked by magnetic DEX-SP-DOX-mAb-1 nanocarriers and magnetic DEX-SP-DOX-1 nanocarriers are shown with blue points in Fig. 8. The results indicate that the higher percentages of cells were marked with the anti-HER2-conjugated magnetic nanocarriers compared with non-conjugated magnetic nanocarriers. Furthermore, translocation and cellular uptake of the targeted nanocarriers differ from those of free antibody ones. According to the images shown in Fig. 8, targeting magnetic nanocarriers demonstrated a greater qualitative amount of translocation and cellular uptake.
3.8 Transmission electron microscopy study TEM images in Fig. 9 were taken to show internalization of targeting nanocarriers into SKBR3 cells through specific cellular uptake. Arrows are used to show the localization of some magnetic nanocarriers within the cells as well as the accumulation of nanocarriers, observed in different regions of the cell.
4. Discussion In recent decades, the use of DOX as a chemotherapeutic drug with wide spectrum of anticancer activities for targeted drug delivery systems based on MNPs has been aroused 18
widespread and reported so well by extended knowledge of its chemical properties (Lu et al., 2012; Sanson et al., 2010; Yang et al., 2011). This study aimed to develop a theranostic system using a cationic polysaccharide, DEX-SP with various desirable properties. The primary amine content of DEX–SP conjugate by TNBS method represented an acceptable value of 1.24 mmol amine/g polymer (Azzam et al., 2002). The synthesis of DOX-loaded DEX-SP magnetic nanocarriers were carried out via ionic gelation method in the presence of TPP as a crosslinker. Such self- assembly method for efficient entrapment of super paramagnetic iron oxide and DOX
has been previously
reported in respectively
poly(ethylene glycol)-block-poly(ɛ-caprolactone) (PEG-PCL) nanomicelles, poly (ethylene oxide)-trimellitic anhydride chloride-folate (PEO-TMA-FA), hydrophobically modified glycol chitosans nanoparticles (Liao et al., 2011; Maeng et al., 2010; Park et al., 2006). Different weigh ratios of DOX to DEX-SP, with or without anti-solvent in the synthesis medium, were used for drug entrapment in nanocarriers. The loading efficiency decreased by increasing DOX to polymer ratio due to limited solubility of drug in polymer solution. The entrapment of DOX in nanocarriers increased in the presence of anti-solvents as a result of solvent removal from polymeric solution in which the drug was dissolved. The zeta potential of the nanocarriers did not change considerably by variation of DOX/polymer ratio, but the hydrodynamic diameter of the nanocarriers increased by increase of DOX to DEX-SP ratio. The polydispersity index was adequately low (
