Anticancer Activity of Tamoxifen Loaded Tyrosine Decorated Biocompatible Fe3O4 Magnetic Nanoparticles Against Breast Cancer Cell Lines
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In this work we reported the synthesis of tamoxifen (TMX) loaded l-tyrosine natural amino acids (Tyr) modified Fe3O4 magnetic nanoparticles. Tyr, which was containing phenol groups was selected to study their effects on biocompatibility, loading capacity and release profile of TMX. TMX loaded Tyr modified Fe3O4 magnetic nanoparticles (F@Tyr@TMX NPs) were characterized by X-ray diffraction, thermo gravimetric analysis, Fourier transform infrared spectroscopy, vibrating sample magnetometer, dynamic light scattering and transmission electron microscopy techniques. The results showed that the ζ-potential of F@Tyr@TMX NPs was about − 12.8 mV and the average size was 22.19 ± 3.58 [mean ± SD (n = 50)] nm. The loading capacity of 11.34 ± 0.09% and encapsulation efficiency of 51.21 ± 0.41%. Additionally, hemolysis test and MTT assays on HEK-293 were performed for determination of biocompatibility of F@Tyr@TMX NPs. Finally, the anticancer activity of F@Tyr@TMX NPs studied on MCF-7 breast cancer cell lines. The results indicate that these as prepared magnetic nanoparticles are suitable for delivery of TMX and even other hydrophobic drugs.
KeywordsMagnetic nanoparticles l-Tyrosine Cancer Drug delivery Tamoxifen Hemolysis
Cancer remains one of the world’s most saddening illnesses, with more than 10 million new cases every year . Though, mortality has reduced in the recent years owing to better understanding of tumor biology and developed diagnostic and treatments strategies. Current cancer cures contain surgical intervention, radiation and chemotherapeutic agents, which often also kill normal cells and cause harmfulness to the patient.
Tamoxifen (TMX) is one of the most extensively used hormonal treatments for breast cancer and has been agreed for the inhibition of breast cancer in high-risk women , nevertheless it encourages dose-dependent side effects such as increased blood clotting, retinopathy and corneal opacities . Also, free drugs may diffuse nonspecifically, a nanosystem can escape into the tumor tissues via the leaky vessels by the EPR effect . Hence, use of biocompatible targeted nanosystems for efficient delivery of TMX can provide maximal therapeutic effects with minimal negligent side effects.
Among the nanosystems that used for cancer therapy [5, 6], iron oxide magnetic nanoparticles have attracted considerable attention in drug delivery systems for cancer research [7, 8], because of their biocompatibility, physical properties, low toxicity, stability, easy of surface functionalization and magnetic properties. Moreover, magnetic nanoparticles have been used for various applications such as catalytic processes [9, 10, 11], cell labeling , hyperthermia therapy , contrast agents for magnetic resonance imaging (MRI)  and drug delivery . Unfortunately, bare magnetic nanoparticles show somewhat aggregation and the coagulation of the nanoparticles during usage is frequently unavoidable . In addition, may also interact with plasma proteins upon intravenous injection. Furthermore, for successful use of magnetic nanoparticles for industrial and biomedical applications it is main requisite to surface specific coating and modification . In this regard, a various group of organic and inorganic materials, such as cellulose , chitosan , polyethylene glycol , graphene , Albumin , dopamine  and amino acids [21, 22] are utilized in these coating processes. Amino acids is one of the molecules that can be applied for the surface stabilization and coating of Fe3O4 nanoparticles due to inexpensive, nontoxic and biocompatible features. In recent years, diverse amino acids have been applied to coatings of Fe3O4 nanoparticles [21, 23, 24, 25, 26, 27]. We have become interested in the development of amino acids coated iron oxide magnetic nanoparticles for biological applications [21, 22].
l-tyrosine (Tyr) is an aromatic and hydrophilic amino acid, can be found in a variety of proteins, and is mainly rich in casein milk protein, molecules containing phenol groups. This supplement is thought to be particularly helpful in a number of processes within the body, including influencing the production of a number of neurotransmitters. Tyr is a non-essential amino acid, is the material of a variety of body products, tyrosine is converted in vivo to a variety of biological substances by different metabolic pathways, such as dopamine, epinephrine, thyroxine, and melanin poppy (opium) of papaverine.
In continuation of our interest in preparation and characterization of drug delivery systems, herein Tyr coated Fe3O4 nanoparticles (F@Tyr NPs) were synthesized and the loading capacity and release profile of the drug from the nanoparticles was evaluated. Furthermore, biocompatibility study and anticancer effect of these nanoparticles investigated.
2 Materials and Methods
l-tyrosine, and FeCl3·6H2O, FeCl2·4H2O were purchased from Merck (Darmstadt, Germany). All other chemicals and solvents were from general lab or HPLC grades, as needed, and were purchased from Emertat Chimi Company (Tehran, Iran). The chemicals were used without any purifications.
2.2 Synthesis of Bare Fe3O4
Bare Fe3O4 was prepared using controlled co-precipitation method as reported before . An aqueous solution (150 mL in deionized water) of FeCl3·6H2O (1.1 g) and FeCl2·4H2O (0.4 g), in the molar ratio 2Fe (III):1Fe (II) prepared and kept at a constant temperature of 60 °C for 15 min under vigorous stirring. Then under vigorous stirring and N2 gas a solution of ammonium hydroxide [20 mL NH4OH (25%)] was added till the pH was reached to ∼ 11 at which a black suspension was formed. This suspension was then stirred at 60 °C for 2 h, under vigorous stirring and N2 gas. Fe3O4 nanoparticles, were separated from the aqueous solution by external magnet, washed with deionized water several times then dried in a vacuum oven overnight.
2.3 Synthesis of l-Tyrosine Coated Fe3O4 Nanoparticles (F@Tyr NPs)
Tyr coated Fe3O4 (nanocarriers) were prepared using controlled co-precipitation one-pot method as reported before . F@Tyr NPs were synthesized by the in-situ, one pot synthesis method. To an aqueous solution (150 mL in deionized water) of a mixture of FeCl3·6H2O (1.1 g) and FeCl2·4H2O (0.4 g), Tyr solution [0.016 mol (2.89 g)] in the molar ratio 2Fe (III):1Fe (II):4 AAs was added and kept at a constant temperature of 60 °C for 15 min under vigorous stirring. Then under vigorous stirring and N2 gas a solution of ammonium hydroxide [20 mL NH4OH (25%)] was added till the pH was raised to ∼11 at which a black suspension was formed. This suspension was then stirred at 60 °C for 6 h, under vigorous stirring and N2 gas. F@Tyr NPs, were separated from the aqueous solution by an external magnet and washed with deionized water several times, then washed with deionized water and then dried in a vacuum oven overnight.
2.4 Preparation of TMX-Loaded Nanocarriers (F@Tyr@TMX NPs)
10 mg of F@Tyr NPs were dispersed in 2.8 mL of distilled water under stirring at 400 rpm. TMX (2.5 mg) was dissolved in 2 mL of ethanol and added dropwise to the F@Tyr NPs suspension. The mixture was stirred for 24 h under the dark condition at room temperature. F@Tyr@TMX NPs (TMX-loaded nanocarriers) were collected by an external magnet and vacuum dried for 24 h at room temperature.
2.5.1 Determination of Particle Size
The morphology and size of the samples was confirmed by the transmission electron microscopy (TEM; Cambridge 360-1990 Stereo Scan Instrument-EDS).
2.5.2 Colloidal Stability
ζ-Potential was assigned by dynamic light-scattering method (DLS) using a nano/zetasizer (Malvern Instruments, Worcestershire, UK, model Nano ZS).
2.5.3 FTIR Analysis
Fourier transform infrared (FTIR) spectra were collected using Bruker Tensor 27 (Biotage, Germany) FT-IR spectrometer.
2.5.4 Thermal Analysis
The thermal stability was determined by thermogravimetric analysis (TGA, Linseis Instruments model, STA PT 1000, USA). The TGA thermograms were recorded at a heating rate of 10 °C min. in the temperature range of 30–500 °C under nitrogen atmosphere.
2.5.5 Structure Characterization
The X-ray diffraction (XRD) patterns were recorded with a Bruker AXS model D8 Advance diffractometer using Cu Ka radiation (k = 1.542 A) with the Bragg angle ranging from 20° to 80°.
2.5.6 Magnetic Properties
Vibrating sample magnetometer (VSM) analysis was done by Meghnatis Daneshpaghoh Co, Kashan, Iran.
2.5.7 Determination of Loading Efficiency
2.6 Drug Release Study
This study was performed to estimate the release of TMX from nanocarriers. In brief, 2.0 mg of F@Tyr@TMX NPs was dispersed in 2.0 mL PBS containing 3% (v/v) Tween 80 and the follow-on suspension was sited within a dialysis bag (Mw 12 kDa) and incubated at 37 °C while immersed in 20 mL of PBS. Then, at predetermined time intervals, 2 mL of the dialysate was taken out and replaced by 2 mL new buffer/Tween solution. The concentration of TMX in the dialysate was measured using UV–Vis spectroscopy at 278 nm.
2.7 Biocompatibility of AAs Coated IONPs
2.7.1 Hemolysis Assay
2.7.2 Cell Viability Assay
The viability of HEK-293 cell lines in the presence of nanocarrier suspensions was assessed relative to the cells in the control experiment (without nanocarrier suspensions). The cells were seeded into a 96-well plate at densities of 1 × 104 cells per well for 24 h. Then the nanocarrier suspensions of six various concentrations ranging from 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 mg/mL were added to cells and incubated for 72 h. After the incubation time, the medium was removed and washed with PBS. Then, MTT solution was added to each well followed by 4 h of incubation at 37 °C, subsequently, the medium was removed and violet formazan crystals were solubilized with DMSO (100 µL). After mild shaking for 15 min, the absorbance ratio of the number of surviving cells in F@Tyr NPs treated samples was compared with the control.
2.8 In Vitro Anticancer Activity
In vitro anticancer activity was determined via MTT assay. Briefly, the cells were cultured in each well of 96-well plates and after 24 h time periods, to allow attachment of the cell to the wells, different concentrations of the materials were treated to the cells. In this context, different concentrations (6.75, 13.5, 27, 54, 108, and 216 µM) of free TMX and F@Tyr@TMX NPs were treated to cells and incubated for 72 h. After the incubation time, the medium was removed and washed with PBS. Then, MTT solution was added to each well followed by 4 h of incubation at 37 °C, subsequently, the medium was removed and violet formazan crystals were solubilized with DMSO (100 µL). After mild shaking for 15 min, the absorbance ratio of the number of surviving cells in free TMX and F@Tyr@TMX NPs treated samples were compared with the control.
3 Results and Discussion
3.1 Characterization of Size and Morphology
The surface of iron oxide magnetic nanoparticles was modified with Tyr by a simple, green and one pot in-situ method. Then, the TMX were physically loaded into the nanocarriers.
Figure 1 shows the TEM studies of F@Tyr@TMX NPs. As shown in the images, nanoparticles with Tyr coatings are all dispersed and have a globular shape without noticeable variation on the morphology. As shown in the TEM image, F@Tyr@TMX NPs are dispersed, have a narrow size distribution with the average size of 22.19 ± 3.58 [mean ± SD (n = 50)] nm.
3.2 Colloidal Stability
The surface charge can mainly affect the stability of the F@Tyr@TMX NPs. Figure 2 shows the ζ-potential of bare Fe3O4, F@Tyr NPs and F@Tyr@TMX NPs. The presence Tyr and TMX on bare Fe3O4 surface was confirmed with the change of ζ-potential. As seen in the Fig. 2 the ζ-potential of bare Fe3O4 was about − 10.1 mV, after functionalized with Tyr the ζ-potential of F@Tyr NPs changed to − 28.2 mV. Also, following the loading of TMX, the ζ-potential of NPs shifted to positive charge (− 12.8 mV). Surface charge is essential in decisive whether the nanoparticles will cluster in blood flow or will stick to or interact with oppositely charged cell membrane. The plasma and blood cells regularly had a negative charge; nanoparticles with slight negative surface charge can decrease nonspecific interaction with these components through electrostatic interactions .
3.3 FTIR Analysis
Figure 3 compares the FTIR spectra of bare Fe3O4, pure Tyr, F@Tyr NPs, pure TMX and F@Tyr@TMX NPs. The presence of the Tyr coating on the Fe3O4 nanoparticles was clearly obvious from the comparison of FTIR spectra of bare Fe3O4 with that of the Tyr coated Fe3O4. The Tyr coated Fe3O4 (F@Tyr NPs) exhibited all the IR absorbance peaks characteristic to the pure Tyr. In F@Tyr NPs the C=O and C–O stretching vibrations can be seen at ~ 1613 and ~ 1362 cm−1 respectively that it was obviously changed with pure Tyr peaks. The weak peaks in 2919 cm−1 area can be attributed to the C–H stretching of methyl or methylene groups that this peak not be seen in bare Fe3O4 and N–H stretching vibration overlaps with O–H stretching at ~ 3400 cm−1. Also, the absorption peaks at 1613 cm−1 which are overlapped with asymmetric COO− stretching due to the N–H bending of amine groups. F@Tyr NPs also exhibit a strong peak at 610 and 440 cm−1 which are assigned to the Fe–O vibration, establishing a covalent bond between covering Tyr and the surface of the Fe3O4 rather than physical absorption. Compared to pure Tyr, shortening of the carboxyl group’s peak is due to interaction with OH groups on the surface of the Fe3O4 [24, 30].
FTIR spectrum of TMX shows characteristic absorption bands at 3027 cm−1 (=C–H stretching), 1507 and 1477(C=C ring stretching) and 3180 cm−1 (–NH2).
FTIR spectrum of the F@Tyr@TMX NPs established peaks related to TMX, at: 1724 cm−1 due to ketonic group, 1588 cm−1 due to amine (N–H bend), and 1384 cm−1 due to methyl group, in addition to Fe3O4 related peaks, at: a strong peak at 613 and 444 cm−1 which are assigned to the Fe–O vibration, which are consistent with the previous reports and published literature .
3.4 Thermal Analysis
Figure 4 shows the thermogravimetric analysis (TGA) and differential thermal analysis (DTA) curves of the F@Tyr NPs and F@Tyr@TMX NPs. A part from this weight loss, the coated sample shows a weight loss of 7.5% corresponding to loss of Tyr which is coated on the nanoparticles. After TMX loading, the F@Tyr@TMX NPs exhibit a much higher weight loss, which is consistent with the increase in organic content of the nanoparticles upon loading of TMX.
3.5 Structure Characterization
F@Tyr@TMX NPs diffractograms obtained by X-ray diffraction (XRD) are presented in Fig. 5. The position and relative intensity of all characteristic diffraction peaks of the F@Tyr@TMX NPs strictly matched those of the standard Fe3O4 nanoparticles, indicating the successful synthesis of the nanoparticles.
The peaks shown in F@Tyr@TMX NPs spectrums are at 2θ equals to 18.49°, 30.28°, 35.62°, 43.26°, 54.12°, 57.36°, and 62.96° which are accorded well to the characteristic peaks and their relative intensities of spinel structure of magnetite corresponding to the (111), (220), (311), (400), (422), (511), and (440) (hkl) planes (JCPDS 019-0629).
3.6 Magnetic Properties
The magnetic properties of bare Fe3O4, F@Tyr NPs and F@Tyr@TMX NPs are studied by measuring magnetization as a function of field, and obtained results [saturation magnetization (Ms) of bare Fe3O4, F@Tyr NPs and F@Tyr@TMX NPs] are shown in Fig. 6. The magnetization of samples were recorded in an applied magnetic field of − 10,000 ≤ H (Oe) ≤ 10,000 at room temperature. The decrease of Ms for Tyr coated Fe3O4 rather than bare Fe3O4 can be clarified by the coating layer on the surface of Fe3O4 nanoparticles . And furthermore the decrease of Ms for F@Tyr@TMX NPs rather than F@Tyr NPs can be clarified by the loading of TMX on the F@Tyr NPs.
3.7 TMX Loading and Release Studies
TMX was used as an anticancer drug to test the loading and controlled release behaviors of TMX and the capability for drug adsorption by Tyr coated Fe3O4 as a nanocarriers. The drug loading ratio (DL) and efficiency of entrapment (EE) percentages of TMX in F@Tyr@TMX NPs were 11.34 and 51.21%, respectively. The cumulative release profiles of TMX is also shown in Fig. 7. In F@Tyr@TMX NPs up to 40% of TMX was released from the nanoparticles in physiological conditions (pH 7.4) after 72 h, whereas under acidic conditions (pH 5.5), about 70% of the loaded TMX was released.
3.8 Biocompatibility of Tyr Coated Fe3O4
To determine the biocompatibility of the Tyr Coated Fe3O4, the nanoparticles should have blood compatibility with the minimal value of hemolytic effect on the human red blood cells (HRBCs). Hemolysis assay was useful to estimate the blood adaptability of F@Tyr NPs. As expected, negligible effect of F@Tyr NPs is obvious in the experiment concentration. The hemolytic activity of the specimens was further quantitatively specified by evaluating the supernatant absorbance at 540 nm (hemoglobin) with UV–Vis spectroscopy. At a high concentration, less than 3.2% hemolytic activity was observed each of the F@Tyr NPs. Consequently, the negligible hemolytic activity of F@Tyr NPs as a nanocarriers confirmed the great biocompatibility of HRBCs, which are desirable for biological utilization especially in drug delivery.
Furthermore, for biomedical usages, toxicity is a critical aspect to consider when evaluating their potential. For drug delivery F@Tyr NPs are intentionally engineered to interact with cells, it is essential to ensure that these enhancements are not causing any adverse effect. The cytotoxicity study of F@Tyr NPs was done on HEK-293 cell lines with different concentrations and the obtained data are shown in the Fig. 8. The cell lines were incubated with nanoparticles for 72 h with concentrations of from 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 mg/mL in a 5% CO2 atmosphere. Figure 8 compares the cell viability as a function of bare and F@Tyr NPs. MTT assays establish no inhibitive effects of F@Tyr NPs against cell lines growth. The outcomes show that the viability of HEK-293 cell lines is not affected by the presence of F@Tyr NPs, suggesting that nanoparticles are highly biocompatible and do not possess a toxic effect.
3.9 In Vitro Anticancer Activity
The in vitro anticancer effects of free TMX, F@Tyr NPs and F@Tyr@TMX NPs against human breast adenocarcinoma (MCF7) were estimated using a MTT assay (Fig. 9). The data exhibit that cell toxicity is directly commensurate to TMX concentration. In the TMX concentration range of 6.75–216 µM, the anticancer activities of the F@Tyr@TMX NPs are upper than the activity of free TMX (Fig. 9). Also the results of this assessment show no toxicity for bare Fe3O4 in various concentrations. These studies specify that F@Tyr@TMX NPs have a very remarkable anticancer effect, for breast cancer cell lines.
In this study, we synthesized F@Tyr NPs using chemical precipitation method to provide biocompatibility and biofunctionalization. The synthesized F@Tyr NPs were loaded with the chemotherapeutic agent, TMX. Both nanoparticles and nanoparticles loaded with TMX were characterized to know their physicochemical characteristics. Hemolysis assay and cytotoxicity studies on HEK-293 cell lines show that as prepared iron oxide nanoparticles are biocompatible. From the cellular cytotoxicity study, F@Tyr@TMX NPs exhibited more cytotoxic effect than that of free TMX. The main therapeutic advantage of drug loaded nanoparticles in magnetically targeted drug delivery is expected to be more pronounced in in vivo studies where the efficacy of free drug is negligible. Thus, future studies will be focused on optimizing the delivery of amino acid coated Fe3O4 nanoparticles in vivo using tumor-model animals.
This work has been supported financially by Faculty of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran (Grant No, A-12-966-10).
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
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