Comparison, synthesis and evaluation of anticancer drug-loaded polymeric nanoparticles on breast cancer cell lines

Abstract

Breast cancer is a major form of cancer, with a high mortality rate in women. It is crucial to achieve more efficient and safe anticancer drugs. Recent developments in medical nanotechnology have resulted in novel advances in cancer drug delivery. Cisplatin, doxorubicin, and 5-fluorouracil are three important anti-cancer drugs which have poor water-solubility. In this study, we used cisplatin, doxorubicin, and 5-fluorouracil-loaded polycaprolactone-polyethylene glycol (PCL-PEG) nanoparticles to improve the stability and solubility of molecules in drug delivery systems. The nanoparticles were prepared by a double emulsion method and characterized with Fourier Transform Infrared (FTIR) spectroscopy and Hydrogen-1 nuclear magnetic resonance (¹HNMR). Cells were treated with equal concentrations of cisplatin, doxorubicin and 5-fluorouracil-loaded PCL-PEG nanoparticles, and free cisplatin, doxorubicin and 5-fluorouracil. The 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide (MTT) assay confirmed that cisplatin, doxorubicin, and 5-fluorouracil-loaded PCL-PEG nanoparticles enhanced cytotoxicity and drug delivery in T47D and MCF7 breast cancer cells. However, the IC50 value of doxorubicin was lower than the IC50 values of both cisplatin and 5-fluorouracil, where the difference was statistically considered significant (p˂0.05). However, the IC50 value of all drugs on T47D were lower than those on MCF7.

Keywords::

• anti-cancer drugs
• breast cancer
• cisplatin
• doxorubicin
• 5-Fluorouracil

Introduction

The synthesis, characterization, engineering, and use of materials and devices of 100 nanometers or less is called nanotechnology (Wang et al. 2012). In cancer therapy, nanotechnology has become a potential and powerful application for the development and progress of nanoparticles as drug delivery systems. Nowadays, nanoparticles have become extremely attractive and applicable for their applications in the fields of medicine and biology (Xu et al. 2007, Mahapatro and Singh 2011). Nanoparticles are spherical and solid structures prepared from synthetic or natural polymers, and range around 100 nm in size. The delivery of a large group of drugs, like vaccines, biological macromolecules, hydrophobic small drugs, and hydrophilic small drugs, can be managed using nanoparticles . Targeted delivery to particular organs or cells can also be enhanced using nanoparticles (Kumari et al. 2010, Hillaireau and Couvreur 2009, Danhier et al. 2012). Targeted anticancer drug delivery has the advantages of few side effects and low toxicity, which enhance therapeutic efficacy rates for cancer chemotherapy (Farokhzad and Langer 2009, Bailey and Berkland 2009). Among a range of drug carriers, polymer nanoparticles have been more popular, because of their potential and ability for targeted and preferential delivery of drugs to cancer tissues (Ghosh et al. 2008, Moghimi et al. 2010). Nanoparticles with a hydrophilic shell, such as polymer micelles with polyethylene glycol (PEG) outer layers, have “stealth” properties, and can thus escape detection by the reticuloendothelial system (RES). They have a delayed circulatory time in the bloodstream, and have enhanced permeation and retention (EPR) effects, which allow their accumulation in solid tumors (Torchilin 2007, Bogdanov et al. 1999). The development of a molecular target that will adjust mechanisms of several signaling pathways would be appropriate for anticancer therapy. Cisplatin, doxorubicin, and 5-fluorouracil are three main and important drugs in chemotherapy, which nowadays attract global attention because of their powerful potential in cancer therapy. Cisplatin is one of the chemotherapeutic drugs which are well-known to crosslink DNA molecules in different ways to hinder cell division via mitosis. The main disadvantage of cisplatin is its toxicity in healthy tissues after the full therapeutic exploitation (Rosenberg 1985). On other hand, doxorubicin, an anthracycline antibiotic, is one of the most broadly used anticancer agents and shows high anticancer activity. The main disadvantage of doxorubicin is its dependent cardio toxicity and myelosuppression (Misra and Sahoo 2010). 5-fluorouracil is a chemotherapeutic drug, which is known as a pyrimidine analog. In this study, we compare the cell toxicity of cisplatin and doxorubicin in free and polycaprolactone-polyethylene glycol (PCL-PEG)-encapsulated forms.

Materials and methods

PCL (1000), PEG (4000), Polyvinyl alcohol (PVA), stannous octoate (Sn (Oct) 2: stannous 2-ethylhexanoate) and dichloromethane (DCM) were purchased from Sigma-Aldrich (USA). Scanning electron microscopy (SEM) measurements were recorded using the KYKY model EM3200. The drug-loading (DL) capacity was determined using a UV-Vis 2550 spectrometer (Shimadzu). Infrared spectra were recorded at room temperature with Fourier transform infrared spectroscopy (FTIR, Perkin Elmer Series). The Hydrogen-1 nuclear magnetic resonance spectra (¹H NMR) were recorded at room temperature (RT) with a Brucker DRX 300 spectrometer operating at 300.13 MHz. The organic phase was vaporized using a rotary (Rotary Evaporator, Heidolph Instruments, Hei-VAP series). A homogenizer (Silent Crusher M, Heidolph Instruments GmbH, Schwabach, Germany) was used to homogenize the samples. MCF7 and T47D breast cell lines were purchased from the Pasteur Institute of Iran. Trypsin-EDTA, RPMI-1640, and fetal bovine serum (FBS) were from Gibco, Invitrogen (UK). We also purchased 0.08 mg penicillin-G (Serva co, Germany), and 2 mg sodium bicarbonate (Merck, Germany), Sigma-Aldrich (USA) supplied 0.2 mg amphotericin B, 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Invitrogen, UK), and 50 mg streptomycin (Merck, Germany) which were was supplemented with one liter of RPMI 1640, MTT (3(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide), and DMSO (dimethyl sulfoxide).

Cell culture and cell line

MCF7 and T47D breast cell lines were cultured in RPMI1640 complemented with 0.08 mg/ml of streptomycin (Merck, Germany), 0.05 mg/ml penicillin-G, 10% heat-inactivated FBS, and 2 mg/ml sodium bicarbonate, and the cells were grown at 37°C in an incubator with 55% humidity and 5% CO2.

Synthesis and characterization of PCL (1000)-PEG (4000)-PCL (1000) triblock copolymers

The PCL (1000)-PEG (4000)-PCL (1000) triblock copolymers were synthesized by an already reported method, with minor modifications (Feng et al. 2012). The chemical structure is shown in Figure 1A. Briefly, ring- opening polymerization of ε-caprolactone was induced by using PEG as the initiator, and Tin(II) 2-ethylhexanoate or stannous octoate (Sn (Oct)2) as the catalyst. 3gr ethylene glycol and 7.4 gr ε-caprolactone were heated in a dry three-necked flask and under vacuum for 10 min, to a temperature of 130°C and under nitrogen atmosphere, until melting was completed (Figure 2A and B). The molar proportion of ethylene glycol and ε-caprolactone was 1/2.5. Then, 0.05% (w/w) (about 40λ) of Sn (Oct)₂ was added into a three-necked flask under a nitrogen atmosphere, and the temperature of the reaction mixture was increased to 180°C. A heater device was used to reach to 180°C, with the stirring condition continued for 6 h, and polymerization was carried out (Akbarzadeh et al. 2012, Valizadeh et al. 2012, Akbarzadeh et al. 2013, Mollazade et al. 2013, Nejati-Koshki et al. 2013).

Figure 1. Chemical structure of (A) PCL-PEG, (B) Cisplatin, (C) Doxorubicin, and (D) 5-Fluorouracil.

Figure 2. (A) Schematic structure of three-necked flask under stirrer and heater device, (B) three-necked flask under stirrer and heater device in our laboratory, (C) a firm and milky mixture of polymer, and (D) dissolving polymer in DCM (dichloromethane).

After 12 h of cooling at room temperature, a firm and milky mixture was obtained (Figure 2C). The copolymer was dissolved by dichloromethane (Figure 2D). To achieve precipitation, after 1 h, ice-cold diethyl ether was added under stirring conditions (Rezaei-Sadabady et al. 2013, Fallahzadeh et al. 2010, Ebrahimnezhad et al. 2013, Pourhassan-Moghaddam et al. 2013, Ahmadi et al. 2014, Davaran et al. 2013, Ghasemali et al. 2013, Sadat et al. 2014, Davaran et al. 2014). The solution was precipitated after 24 h. The precipitate was stored in a desiccator until it was used (Rezaei-Sadabady et al. 2013, Fallahzadeh et al. 2010, Ebrahimnezhad et al. 2013, Pourhassan-Moghaddam et al. 2013, Ahmadi et al. 2014, Davaran et al. 2013, Ghasemali et al. 2013, Sadat et al. 2014, Davaran et al. 2014). The morphology of the nanoparticles was examined using SEM. A Brucker DRX 300 MHz spectrometer was used and the ¹H NMR spectra were recorded in CDCI3. The samples were homogenized using a homogenizer (Heidolph Instruments GmbH and Co. KG, SilentCrusher M). The organic phase was evaporated by rotary (Rotary Evaporators, Heidolph Instruments, Hei-VAP Series). The FTIR spectrum was gained from a neat film cast of the chloroform copolymer solution between KBr tablets (Kouhi et al. 2014, Abbasi et al. 2014, Pourhassan-Moghaddam et al. 2014, Hosseininasab et al. 2014, Davoudi et al. 2014, Taheri et al. 2014, Alimirzalu et al. 2014, Eatemadi et al. 2014).

Drug loading

Nowadays, there are different techniques for loading the drug into nanoparticles. Combinations of different techniques were used to prepare for the loading. These were methods such as double emulsion (w/o/w), probe type sonication, and solvent evaporation (Figure 3). PCL-PEG nanoparticles loaded with cisplatin were prepared using the double emulsion method (Karnoosh-Yamchi et al. 2014, Alizadeh et al. 2014, Nejati-Koshki et al. 2014, Ebrahimi et al. 2014, Ghalhar et al. 2014, Daraee et al. 2014, Sadat et al. 2014, Daraee et al. 2014). An aqueous solution of cisplatin (0.4 ml) was added and emulsified in 2 ml of dichloromethane (Karnoosh-Yamchi et al. 2014, Alizadeh et al. 2014, Nejati-Koshki et al. 2014, Ebrahimi et al. 2014, Ghalhar et al. 2014, Daraee et al. 2014, Sadat et al. 2014, Daraee et al. 2014). Thus, 100 mg of the copolymer was emulsified in the mixture, using probe sonication at 10 W for 45 s. The mixture obtained (w/o emulsion) was added to an aqueous solution of sodium cholate (6 ml), and was then probe sonicated at 18 W for 1 min. The mixture obtained (w/o/w emulsion) was gently stirred at room temperature until the evaporation of the organic phase was complete (Nasrabadi et al. 2014, Chung et al. 2014, Fekri Aval et al. 2014, Zohre et al. 2014, Valizadeh et al. 2014, Mellatyar et al. 2014, Dadashzadeh et al. 2014, Rahimzadeh et al. 2014, Badrzadeh et al. 2014, Herizchi et al. 2014, Kafshdooz et al. 2014, Sohrabi et al. 2014, Tozihi et al. 2014, Shafiei et al. 2014, Kordi et al. 2014, Barkhordari et al. 2014, Dadashzadeh et al. 2014, Zare et al. 2014, Kordi et al. 2014, Majidi et al. 2014). To prepare doxorubicin-loaded PEG-PCL, 3 mg of doxorubicin hydrochloride was emulsified in 2 ml of dichloromethane (CH2Cl2) in the presence of a triple molar ratio of triethylamine, at room temperature, and stirred at 400 rpm on a magnetic/heater stirrer for 6 h (Nasrabadi et al. 2014, Chung et al. 2014, Fekri Aval et al. 2014, Zohre et al. 2014, Valizadeh et al. 2014, Mellatyar et al. 2014, Dadashzadeh et al. 2014, Rahimzadeh et al. 2014, Badrzadeh et al. 2014, Herizchi et al. 2014, Kafshdooz et al. 2014, Sohrabi et al. 2014, Tozihi et al. 2014, Shafiei et al. 2014, Kordi et al. 2014, Barkhordari et al. 2014, Dadashzadeh et al. 2014, Zare et al. 2014, Kordi et al. 2014, Majidi et al. 2014). Then, 10 mg of PEG-PCL copolymer was added and emulsified into the mixture obtained, and stirred at 400 rpm until it was completely emulsified. Afterwards, the obtaining solvent was added and emulsified drop wise in 20 mL of distilled water. The mixture was sonicated 16 times at intervals, and the 1-min pulse was turned off for 1 s at 15-s intervals. For solvent evaporation, micelle formation, and removal of the residual CH2Cl2, the beaker was left open to air during the night, and using a rotary evaporator, and the residual solvent was removed. The procedure of preparation of 5-flurouracil was the same as that for the preparation of the doxorubicin-loading method, but we used 10 mg of 5-fluorouroacil and 100 mg of polymer. For the elimination of cisplatin, doxorubicin and 5-fluorouracil aggregates, all solution was filtered with a syringe filter (pore size 0.22 m). All the procedures were carried out under vacuum and in a nitrogen atmosphere.

Figure 3. Preparation of encapsulated drugs. The drugs were loaded in nanoparticles with a combination of different techniques, which are known as w/o/w or water-in-oil-in-water (double emulsion). (A) Water [W], (B) Oil [O], (C) W/O emulsion, (D) W/O/W emulsion, and (E) Schematic structure of encapsulated drugs.

Determination of entrapment efficiency (EE) and drug loading (DL)

The solution obtained in the last step was centrifuged for 30 min at 10,000 rpm. To calculate the encapsulation efficiency (EE) and the drug-loading (DL) rate of the nano particles, for cisplatin, doxorubicin, and 5-fluorouracil, the supernatant was cut off and used for comparison with the total amount of cisplatin, doxorubicin, and 5-fluorouracil. The amount of non-encapsulated cisplatin, doxorubicin, and 5-fluorouracil in the supernatant was calculated by using an ultraviolet 2550 spectrophotometer (Shimadzu), with an absorbance peak at 301 ± 2 (cisplatin), 254 (doxorubicin), and 266 (5-fluorouracil). The following formula was used to measure the percent of cisplatin, doxorubicin and 5-fluorouracil encapsulated in the nanoparticles (Eq. 1), and the rate of DL (Eq. 2).

(1) (2)

Cell lines and culture cmonditions

The human breast cancer cell lines MCF7 and T47D were cultured in a RPMI 1640 culture medium supplemented with 2 mg of sodium bicarbonate, 2 mM L-glutamine, 10% FBS, 0.2 mg of amphotericin B, penicillin-G (80 mg/mL), and streptomycin (50 mg/mL) at 37°C in a 5% humidified CO2 incubator.

Cell viability and MTT-based cytotoxicity test

Cells were exposed to free cisplatin, doxorubicin, and 5-fluorouracil, and cisplatin, doxorubicin, and 5-fluorouracil-loaded PCL-PEG, during the exponential phase of growth, and cytotoxicity was measured at 24-h after treatment using the MTT assay. First, in a 96-well plate (Costar^® from Corning, NY) 10000 cells/well were seeded and kept for 24 h in the incubator, to promote cell attachment. Then, both T47D and MCF7 cell lines were treated, in triplicate, with different concentrations of free cisplatin, doxorubicin and 5-fluorouracil, and cisplatin, doxorubicin, and 5-fluorouracil-loaded PCL-PEG (0.312–20 μM for doxorubicin, 5–320 μM for cisplatin, and 6–344 μM for 5-fluorouracil), for 24 h. Three controls were used, namely 1% DMSO and PCL-PEG control, for estimation of nanoparticle effect, and a control of the cells alone. After 24 h of drug exposure, the medium was replaced with 200 μl fresh medium for 24 h. After that, the cells were incubated with 50 μL of 2 mg/ml MTT, which was dissolved in PBS, for 4 h. By this method, the plates were covered with aluminum foil. The content of all wells was removed, and 200 μL of pure DMSO and 25 μL of Sorensen's glycine buffer were added to each well. Finally, an ELISA reader (with a reference wavelength of 630 nm) was used to calculate the absorbance measurement at 570 nm.

Results

FTIR spectrum of poly caprolactone 1000- poly ethylene glycol 4000- poly caprolactone 1000 (PCL-PEG-PCL) copolymer

The PCL-PEG-PCL co-polymer nanoparticles were successfully perpetrated by ring-opening copolymerization of PEG and ε-CL (Figure 4). FTIR spectroscopy was done using a Shimadzu spectrophotometer. The FTIR spectrum is compatible with the structure of the presupposed PCL-PEG copolymer. FTIR spectroscopy was used to show the original and main structure of the PCL-PEG copolymer nanoparticle. As illustrated in Figure 4, due to the coupling reaction of –NCO with the –OH group, there is no absorption at 2250–2270 cm^{− 1}, which means that the –NCO groups of hexamethylene diisocyanate disappeared completely. The ester bond appeared at 1721 cm^{− 1} with a strong C = O stretching band. The absorption bands at 1528 cm^{− 1} are attributed to the N–H bending vibrations, which proved the construction of PCL-PEG-PCL triblock copolymers. The terminal hydroxyl groups in the copolymer, from which the PEG homopolymer was removed, was attributed to the appearance of the absorption band at 3509.9 cm^{− 1}. Our FTIR results were compared with other studies, and showed strong similarity of data (Feng et al. 2012). These absorption bands indicated that the PCL-PEG-PCL block copolymer was successfully synthesized (Figures 5, 6).

Figure 4. Synthesis of poly (ε-caprolactone)-poly (ethylene glycol)-poly (ε-caprolactone).

Figure 5. Fourier transform infrared spectra (FTIR) of poly (ε-caprolactone)-poly (ethylene glycol)-poly (ε-caprolactone).

Figure 6. Comparison of FTIR of poly (ethylene glycol) (A), ε-caprolactone (B), and poly (ε-caprolactone)-poly (ethylene glycol)-poly (ε-caprolactone) (C).

¹H NMR spectrum of the PCL- PEG co-polymer

To confirm the basic chemical structure of PCL-PEG-PCL triblock nanoparticles, the ¹H-NMR spectrum was recorded at RT, with a Brucker DRX 300 spectrometer operating at 400 MHz (Figure 7). One of the important features was a large and sharp single peak at 3.65 ppm, corresponding to the methylene groups of the PEG segments. The very weak peak at 4.23 ppm was attributed to the methylene proton of the PEG end unit. In addition, peaks at 4.06, 2.31, 1.65 and 1.38 ppm were corresponding to the methylene protons of OCOCH2CH2CH2CH2–CH2, OCO-CH2–CH2)4, OCOCH2–CH2–CH2–CH2–CH2, and OCOCH2CH2–CH2–CH2CH2 in the PCL segments, respectively. Our data was very similar to the reported spectra (Feng et al. 2012, Jia et al. 2008). All the FTIR and ¹H-NMR results showed that the PCL-PEG-PCL triblock copolymer was synthesized successfully.

Figure 7. ¹HNMR spectrum of poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone).

Entrapment efficiency (EE) and drug loading (DL)

The cisplatin, doxorubicin and 5-fluorouracil content in the drug-loaded nanoparticles were determined using the ultraviolet spectrophotometer at 301 ± 2, 254 and 266 nm, respectively. The DL content and drug EE were calculated based on the Equations 1 and 2, by which the values for the EE for cisplatin, doxorubicin, and 5-fluorouracil were 98.8%, 99.1%, and 98.9%, respectively. The DL for cisplatin, doxorubicin, and 5-fluorouracil were 13.53%, 16.12%, and 14.24%, respectively.

Cell cytotoxicity (MTT assay)

In this study, cell viability was evaluated by the MTT assay, by exposing the T47D and MCF7 cell lines to different concentrations of pure cisplatin, doxorubicin, and 5-fluorouracil, and cisplatin, doxorubicin, and 5-fluorouracil-loaded PCL-PEG, for 24 h. By increasing the amount of drug, the cell toxicity outcome was boosted, which demonstrates how cisplatin, doxorubicin, and 5-fluorouracil are dose-dependent and the cell viability is inversely proportional. The pure cisplatin, doxorubicin and 5-fluorouracil had a cytotoxic effect on T47D and MCF7 cell lines. The values for inhibitory concentration at 50% (IC50), for free cisplatin, doxorubicin, and 5-fluorouracil on T47D, were 70.91, 3.001 and 28.46 for 24 h, respectively. The IC50 values of encapsulated cisplatin, doxorubicin, and 5-fluorouracil on T47D were 50.32, 1.626 and 39.11 for 24 h, respectively (Figures 8, 9, 12, 13, 16, and 17). The IC50 values of free cisplatin, doxorubicin, and 5-fluorouracil on MCF7 were 44.17, 2.286 and 33.65 for 24 h, respectively (Figures 7a–c). The values for IC50 of encapsulated cisplatin, doxorubicin, and 5-fluorouracil on MCF7 were 26.39, 1.23 and 23.79 for 24 h, respectively (Figures 10, 11, 14, 15, 18, and 19).

Figure 8. Cytotoxic effect of free and encapsulated 5-Fluorouracil on T47D over a 24-h exposure.

Figure 9. Normalized MTT assay data for free and encapsulated 5-flourouracil on T47D over a 24-h exposure.

Figure 10. Cytotoxic effect of free and encapsulated 5-flourouracil on MCF7 over 24-h exposure.

Figure 11. Normalized MTT assay data for free and encapsulated 5-flourouracil on MCF7 for 24-h exposure.

Figure 12. Cytotoxic effect of free and encapsulated cisplatin on T47D over a 24-h exposure.

Figure 13. Normalized MTT assay data for free and encapsulated cisplatin on T47D for 24-h Exposure.

Figure 14. Cytotoxic effect of free and encapsulated cisplatin on MCF7 over a 24-h exposure.

Figure 15. Normalized MTT assay data for free and encapsulated cisplatin on MCF7 for 24-h exposure.

Figure 16. Cytotoxic effect of free and encapsulated doxorubicin on T47D over a 24-h exposure.

Figure 17. Normalized MTT assay data for free and encapsulated doxorubicin on T47D for 24-h exposure.

Figure 18. Cytotoxic effect of free and encapsulated doxorubicin on MCF7 over a 24-h exposure.

Figure 19. Normalized MTT assay data for free and encapsulated doxorubicin on MCF7 for 24-h exposure.

PCL-PEG and DMSO showed an absorbance value equivalent to 99% and 98% of control for MCF7, and 98% and 97% for T47D, respectively. It shows that DMSO and PCL-PEG have very low effect on the cells. The time-dependency of this drug was suggested by the lack of similarity between results for different periods. The graph was plotted using PRISM 4. The statistical significances were evaluated by the t-test, and a value of p ˂ 0.05 was considered significant.

Discussion

Breast cancer is the main cause of the high cancer-related mortality rate in women. The delivery of anticancer drugs for cancer therapy may involve direct delivery into the tumor. Nowadays, chemotherapy is the main method for cancer therapy, but it always has many different side effects. While in the treatment of human cancer, chemotherapy has toxic side effects in healthy and cancerous tissues, nanotechnology attempts to resolve these problems by encapsulating or loading drugs in material which can deliver drugs directly to cancerous tissues (Eatemadi et al. 2014, Eatemadi et al. 2014, Daraee et al. 2014, Daraee et al. 2014, Seidi et al. 2014). By synthesis of a core-shell nanoparticle formulation of chemotherapeutic agents, the delivery of an efficient agent to where it is needed can be easier and the efficiency of treatment can be enhanced, and it may also be helpful for the treatment of tumors. By this method, the selective delivery of cisplatin, doxorubicin, and 5-fluorouracil to tumor cells would considerably reduce drug toxicity, and improve their therapeutic index. As discussed above, co-polymers (such as PCL-PEG, PLGA-PEG, and PLA-PEG) promise to be a successful system for the targeted and controlled release of cisplatin, doxorubicin, and 5-fluorouracil, with decreased systemic toxicity, increased therapeutic efficiency, and patient compliance. This study explored the cell toxicity effect of the three main chemotherapeutic drugs (cisplatin, doxorubicin, and 5-fluorouracil) on two cell lines attributed to breast cancer (T47D and MCF7) to obtain appropriate data for comparison. The results suggest that drug-loaded PCL-PEG is a suitable candidate for chemotherapy. The entrapment of cisplatin, doxorubicin, and 5-fluorouracil within a nanoparticulate carrier noticeably improved the IC50 and cell toxicity parameters of cisplatin, doxorubicin, and 5-fluorouracil. Also, either free or encapsulated doxorubicin had a cytotoxic effect at a minimum amount on MCF7 and T47D, as compared with free and encapsulated cisplatin and 5-fluorouracil, where the difference was statistically considered significant (p ˂ 0.05). The free and encapsulated cisplatin had higher IC50 values than doxorubicin and 5-fluorouracil, where the difference was statistically considered significant (p ˂ 0.05). Also, all IC50 values of the free and encapsulated drugs in T47D were lower than those for MCF7, and this result suggests that T47D is more sensitive than MCF7, where the difference was statistically considered significant (p ˂ 0.05).

Authors’ contributions

AE conceived of the study and participated in its design and coordination. AA participated in the sequence alignment and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors thank the Department of Medical Nanotechnology, Faculty of Advanced Medical Science of Tabriz University, for all support provided. This work is funded by a 2014 grant by the Drug Applied Research Center, Tabriz University of Medical Sciences. This work is funded by a 2014 grant by the Drug Applied Research Center, Tabriz University of Medical Sciences, and Student Research Committee.

Declaration of interest

The authors have no declaration of interest. The authors alone are responsible for the content and writing of the paper.