Elegant pH-Responsive Nanovehicle for Drug Delivery Based on

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An Elegant pH-Responsive Nanovehicle for Drug Delivery Based on Triazine Dendrimer Modified Magnetic Nanoparticles Amir Landarani-Isfahani, Majid Moghadam, Shima Mohammadi, Maryam Royvaran, Naimeh MoshtaelArani, Saghar Rezaei, Shahram Tangestaninejad, Valiollah Mirkhani, and Iraj Mohammadpoor-Baltork Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00742 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 22, 2017

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Table of Contents Graphic and Synopsis

pH-Responsive Triggered Drug Release

Triazine Dendrimer-Magnetic Nanoparticles Owing to properties of magnetic nanoparticles and elegant three-dimensional macromolecule architectural features, dendrimeric structures have been investigated as nanoscale drug delivery systems. In this paper, a novel magnetic nanocarrier, generation two (G2) triazine dendrimer modified Fe3O4@SiO2 magnetic nanoparticles (MNP-G2), was designed, fabricated and characterized by variety techniques. To demonstrate the potential of MNP-G2, the nanoparticles were loaded with methotrexate (MTX), a proven chemotherapy drug. In vitro experiments exhibited a high drug loading capacity of MTX, the excellent ability for controlled drug release and appreciate biocompatibility.

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An Elegant pH-Responsive Nanovehicle for Drug Delivery Based on Triazine Dendrimer Modified Magnetic Nanoparticles Amir Landarani-Isfahani,†‫ ٭‬Majid Moghadam,†‫ ٭‬Shima Mohammadi,‡ Maryam Royvaran,

‡

Naimeh Moshtael-Arani,§ Saghar Rezaei †, Shahram Tangestaninejad,† Valiollah Mirkhani,† Iraj Mohammadpoor-Baltork† †

Department of Chemistry, University of Isfahan, Isfahan 81746-73441, Iran

‡

Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of

Isfahan, Isfahan 81746-73441, Iran. §

Young Researchers and Elite Club, Kashan Branch, Islamic Azad university, Kashan,

8715998151, Iran *Corresponding authors. Prof. Moghadam and Dr. Landarani-Isfahani are to be contacted at Tel +98 31 37934920; fax: +98 31 36683732. E–mail addresses: [email protected] (Prof. Moghadam); [email protected] (Dr. Landarani)


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ABSTRACT: Owing to properties of magnetic nanoparticles and elegant three-dimensional macromolecule architectural features, dendrimeric structures have been investigated as nanoscale drug delivery systems. In this paper, a novel magnetic nanocarrier, generation two (G2) triazine dendrimer modified Fe3O4@SiO2 magnetic nanoparticles (MNP-G2), was designed, fabricated and characterized by Fourier transform infrared (FT-IR), thermal gravimetric analysis (TGA), vibrating sample magnetometer (VSM), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM) and dynamic light scattering (DLS). The prepared MNP-G2 nanosystem offers a new formulation that combines the unique properties of MNPs and triazine dendrimer as a biocompatible material for biomedical applications. To demonstrate the potential of MNP-G2, the nanoparticles were loaded with methotrexate (MTX), a proven chemotherapy drug. The MTX-loaded MNP-G2 (MNP-G2/MTX) exhibited a high drug loading capacity of MTX and the excellent ability for controlled drug release. The cytotoxicity of MNPG2/MTX using an MTT-based assay and MCF-7, HeLa and Caov-4 cell lines revealed that MNP-G2/MTX was more active against the tumor cells than the free drug in a mildly acidic environment. The results of hemolysis, hemagglutination and coagulation assays confirmed the good blood safety of MNP-G2/MTX. Moreover, the cell uptake and intracellular distribution of MNP-G2/MTX were studied by flow cytometry analysis and confocal laser scanning microscopy (CLSM). This research suggests that MNP-G2/MTX with good biocompatibility and degradability can be selected as an ideal and effective drug carrier in targeted biomedicine studies especially anticancer applications. KEYWORDS: triazine dendrimer; nanoscale; drug delivery systems; anticancer; methotrexate


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1. INTRODUCTION Nano-scaled drug carriers have emerged as a bridge linking nanotechnology and advanced drug delivery, involving nano-scaled materials such as liposomes,1,

2

nanoparticles,3-5 dendrimers,6

polymers,7, 8 micelles,9-11 DNA nanostructures, 12 and virus capsids.13 One of the main objectives in the field of targeted drug delivery is to enhance the therapeutic index of drugs by facilitating their selective drug uptake into target cells.14 MTX, 2,4-diaminoN10-methyl propylglutamic acid, is one of the most widely studied and effective therapeutics agents available to treat breast cancer, acute lymphatic leukemia, osteogenic sarcoma, choriocarcinoma, lung cancer, bladder carcinoma, brain medulloblastoma, primary CNS lymphoma, and chronic myeloid leukemia.15 Although MTX has efficient cytotoxic activity against cancer cells, it suffers from low solubility, dose-limiting systemic toxicity, lack of selectivity, rapid diffusion throughout the body, short half-life in the bloodstream, and drug resistance by target cells that lead to a narrow therapeutic index.16,17 To overcome these difficulties and improve the pharmacokinetic properties, the numerous nanoscaled carriers such as dendrimers,18-20 oligopeptides,21 lipids,22 albumin protein,23 chitosan,24 dextran,25 LDH (layered double hydroxide),26 and iron oxide nanoparticles27 have been used. Magnetite nanoparticles (Fe3O4), as one of the most important magnetic materials, have attracted extensive attention because of their unique physicochemical properties and great potential use in various biomedical applications such as smart carriers for targeted drug delivery,28 a dual imaging probe for cancer,29 the magnetic separation in microbiology, biochemical sensing and so on.30 Furthermore, Fe3O4 nanoparticles induce hyperthermia effects when placed in an alternating magnetic field for tumor thermotherapy.31 As a whole, the magnetic targeting strategy is believed to provide an option in order to guide the accumulation of nanocarriers in tumor


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tissues and facilitate their internalization into tumor cells. Eventually, anti-cancer drugs will be released from nano carrier to cancer cells and further diffuse into the nuclei from cytosol, interrupting DNA replication and, thus, leading to cell death.32 To overcome the instability and aggregation of Fe3O4 nanoparticles, its surface is usually modified with a silica layer, taking advantages of biocompatibility as well as high density of surface functional end groups, and the great ability for connecting to various inorganic, organic or biomolecules.33-35 MNPs functionalized with macromolecules form distinct particulate systems that can pass through cellular barriers and offer organ-specific therapeutic and diagnostic tools.36 Such a capability will open the door toward the design of magnetic carrier that can be used to deliver specific ligands to target organs. In recent years, dendrimers as an elegant class of macromolecules have been extensively utilized as drug delivery platforms by virtue of their unique branched structure, uniform size distribution, aqueous solubility, biocompatibility, and chemically linkable surface multifunctional groups.37 Furthermore, some dendrimer–drug conjugates was reported as safe and smart nanoscale pHresponsive drug delivery systems for cancer therapy.38-42 However, difficult synthesis and purification, long reaction times and use of expensive reagents are often limitations and drawbacks of preparation of dendrimers. One such class of dendrimers is the triazine-based nanostructures. The availability, low cost and ease of synthesis of 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride, CC) as the core reagent makes its adoption even more attractive. In addition, ease of displacement of its chlorine atoms by different primary and secondary amine nucleophiles allows preparing chemically diverse multifunctional dendrimers with high purities.43-45 More recent success in kilogram-scale production43, 46, 47 and preparation of high generation triazine dendrimers,48, 49 have accelerated


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these studies in all areas including host-guest chemistry and chemotherapy.48,

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50, 51

Recently,

triazine dendrimers have been used to deliver genes as well as bioactive macromolecules like DNA and siRNA.52 On the basis of the above information and motivated by the special superparamagnetic properties of Fe3O4@SiO2 core-shell nanoparticles, summarized to MNPs, and high loading capacity of biocompatible triazine dendrimer for encapsulation of drugs, we herein have the opportunity to push our studies toward the synthesis and characterization of MNP-G2 as a novel delivery system for encapsulation of MTX. Herein, we report the development of novel combination of MNP-G2 as the nanoscale drug platform, and the physically encapsulated MTX for cancer drug delivery. The physicochemical properties of MNP-G2 were examined by FT-IR, TGA, VSM, FE-SEM and TEM techniques. The special engineering of functional MNP with triazine dendritic polymer includes large drugloading capacity, pH-dependent drug release for enhance the drug release under intracellular compartments and acidic cancerous conditions, and magnetic cancer targeting. The MTX release characteristics from MNP-G2 were evaluated in vitro under the conditions replicating those necessary for drug delivery. Furthermore, the biocompatibility of MNP-G2 and MNP-G2/MTX was assayed by in vitro hemocompatibility and cytotoxicity tests. Also, the enzymatically degradation of MNP-G2 was examined. The analysis of cellular statistical internalization and cellular localization procedures of MNP-G2/MTX was also determined by CLSM images and flow cytometry.

2. EXPERIMENTAL SECTION


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2.1. Materials Used: All chemicals, solvents, and reagents were purchased from Merck and Sigma-Aldrich Companies and used as received unless otherwise specified. Water used in all experiments was purified by a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA). The 8 kDa molecular weight cut-off cellulose dialysis bag and PBS were acquired from Fisher. The anti-cancer drug MTX hydrochloride was purchased from Pfizer, China. All cell lines were obtained from the National Cell Bank of Iran (NCBI), Pasteur Institute, Tehran, Iran. 2.2. Characterization Techniques: FT-IR spectra were collected using a Jasco FT-IR-6300 instrument in the region of 4000 – 400 cm-1. TGA was carried out using a Mettler TG50 instrument under air flow at a uniform heating rate of 5 °C/min in the range of 30−600 °C. Magnetic properties of the MNPs were recorded as a function of the applied magnetic field sweeping between ±10 kOe at room temperature. All measurements were performed on a vibrating sample magnetometer device (Meghnatis Daghigh Kavir Co., Kashan Kavir, Iran). The FE-SEM image was recorded on Mira II LMU Tescan microscope made in Czech Republic. The TEM micrograph was observed by Philips CM120 with a LaB6 cathode and accelerating voltage of 120 kV. A Shimadzu UV-visible 1650 PC spectrophotometer was used for recording absorption spectra in solution using a cell of 1.0 cm path length. The cellular uptake was examined on flow cytometry (FACS caliber, Becton Dickinson) and confocal laser scanning microscopy (Nikon eclipse Ti microscope). The ultrasonic equipment used for the synthesis of MNPs was a Bandelin electronic ultrasonic bath DT 514 BH-RC 3095, with a working frequency of 40 KHz. The particle size and zeta potential of the nano carrier were determined in aqueous solutions by dynamic light scattering (DLS) using a ZetaSizer Nano Series instrument (Malvern Instruments) at 20 °C.


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2.3. Synthesis of MNP-G2 Triazine Platform: 2.3.1. Preparation of MNPs and MNP-AP. The synthesis of Fe3O4 nanoparticles and their coating with silica shell was carried out according to the reported literature.53 Surface modification of Fe3O4@SiO2 (MNP) was performed with 3-aminopropyltriethoxysilane (APTES). In a round-bottomed flask equipped with a condenser and a mechanical stirrer, 4.5 g of MNP nanoparticles is ultrasonically dispersed in a solution containing 100 mL dry toluene. Then, APTES (5 mL) is added drop-wise into the bottle and the mixture was stirred with a mechanical stirrer at 2000 rpm at 100 °C for 24 h. After hydrolyzing, the nanoparticles are collected by an external magnet and washed with toluene to remove the unreacted starting materials, then dried under vacuum conditions at room temperature. Thus, SiO2-coated Fe3O4 nanoparticles functionalized by –NH2 groups are obtained. 2.3.2. Preparation of MNP-CC1. The MNP-AP (2 g, 0.99 mmol) was added to a solution of cyanuric chloride (CC, 1.85 g, 10 mmol) and diisopropylethylamine (DIPEA, 10 mmol, 1.7 mL) in tetrahydrofuran (THF, 10 mL). The reaction mixture was shaken overnight at 0 °C. The solid material was separated by an external magnet, washed with hot THF to remove the unreacted starting materials and then dried in a vacuum oven at 50 °C. 2.3.3. Preparation of Fe3O4@SiO2-Supported G1 Triazine Dendritic Polymer (MNP-G1). Bis(3-aminopropyl)amine (8.11 mmol, 1 mL) and DIPEA (8.11 mmol, 1.4 mL) were added to a slurry of MNP-CC1 (1 g) in DMF (12 mL). The reaction mixture was stirred at 80 °C for 16 h. The nanoparticles were separated by a magnet, washed with hot ethanol and then dried under vacuum at 50 °C. 2.3.4. Preparation of MNP-CC2. MNP-G1 (1 g, 0.45 mmol) was added to a solution of CC (1.66 g, 9 mmol) and DIPEA (9 mmol, 1.56 mL) in THF (20 mL). The reaction mixture was


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agitated at room temperature for 16 h. The obtained nanoparticles were separated from the mixture by a magnet and washing repeated with hot THF. Finally, the MNP-CC2 was dried under vacuum conditions at 50 °C. 2.3.5. Preparation of Fe3O4@SiO2-Supported G2 Triazine Dendritic Polymer (MNP-G2). To a slurry of MNP-CC2 (1 g, 0.36 mmol) in DMF (20 mL) was added bis(3-aminopropyl)amine (9.36 mmol, 1.14 mL) and DIPEA (9.36 mmol, 1.61 mL). The reaction mixture was stirred at 80°C for 16 h. After the completion of reaction, the resulting was separated, washed with hot THF and then dried in a vacuum oven at 50 °C. Therefore, MNP-G2 prepared in this way can serve as the novel multifunctional platform for loading drug. 2.4. In Vitro Study. All in vitro experiments were performed at least three times, using three replicate samples for each formulation concentration tested. 2.4.1. Drug Loading and Release. The loading content of MTX was achieved as follows: 150 µL of MNP-G2 dispersed in water (0.13 mg.mL−1) was incubated with 2.5 mL different concentrations of MTX (0.025, 0.03, 0.04, 0.05 mg.mL−1) for 24 h under shaking at room temperature until MTX concentration in the solution stabilized. Then, the nanoparticle was separated by an external magnetic field. To remove the excess drug, nanoparticles was further rinsed several times with water and then separated by magnetic separation. All supernatants containing the unloaded MTX were collected. The amount of unloaded MTX was determined by measuring the UV absorbance at 304 nm (the characteristic absorbance of MTX) relative to a calibration curve recorded under identical conditions, allowing the drug loading efficiency to be estimated. To investigate the release of MTX from synthesized MNP-G2, a dialysis process was carried out. Briefly, about 0.002 g of MTX-G2 was dispersed in 5 mL of PBS. The obtained suspension was


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sealed in a dialysis bag immersed in 50 mL of PBS buffer with two different pH values of 7.4 and 5.5

24, 54, 55

at the physiological temperature of 37 °C. After shaking for different times, the

nanocarier was separated by a magnet and washed with PBS. At selected intervals, 1 mL of the solution outside dialysis bag was replaced by the same volume of fresh buffer solution for analysis. The amount of released MTX from MNP-G2 was determined by recording UV absorbance of MTX in the supernatant at 304 nm.

2.4.2. Hemolysis of Red Blood Cells (RBCs) by Nanoparticles. The hemolytic activity of MNP-G2 and MNP-G2/MTX was assayed. 5 mL of fresh and heparinized human blood was centrifuged at 4500 rpm for 10 min to acquire RBCs. The cell pellet as RBCs was washed with cold PBS by centrifugation at 4500 rpm for 10 min. This washing step was repeated for three times and finally, RBCs re-suspended in PBS to reach a concentration of 1.5% (v/v). For hemolysis test, 380 µl of the diluted RBC suspension was mixed with 20 µl of different concentrations (5, 10, 50, 100, 200 µg.mL-1) of MNP-G2 and MNP-G2/MTX dispersed in PBS, separately. After incubation for 30 min at 37 °C, samples were centrifuged at 4000 rpm for 5 min. Finally, 100 µl aliquots of each supernatant were diluted with 900 µl of PBS, and the absorbance of the supernatants measured by a UV-vis spectrophotometer at 576 nm. About 0.2% Triton X-100 was regarded as the positive control (100% lysis) and PBS was regarded as the negative control (0% lysis). 2.4.3. Cell Cultures and In Vitro Cytotoxicity Assays. Two cell lines including MCF-7, HeLa and Coav-4 cancer cells and normal HBL-100 cells were seeded into a 96-well plate at density of 104 cells per well in 200 µL of RPMI-1640 medium and incubated overnight. Then, 10 µL of the corresponding medium containing various concentrations of MNP-G2, free MTX and MNP-


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G2/MTX (5, 10, 50, 100, 200 µg.mL-1) was added to each well and treated for 24 h and 48 h, respectively. After incubation, cell viability was measured by the MTT assay. Briefly, after the cells were washed with PBS for three times, 10 µL of MTT (0.5 mg.mL−1 in PBS) was added to each well. Following a 4-h incubation period, the supernatant was removed and formazan crystals were solubilized in 150 µL of DMSO by shaking for 10 min. The cell viability was quantified spectrophotometrically at 492 nm using a microplate reader (Stat Fax-3200, AWARENESS, Palm City, USA). In addition, to assay the effect of external magnetic field on cytotoxicity, MTT test was performed on cancer and normal cell lines incubated with MNP-G2 and MNP-G2/MTX in different concentration (5, 10, 50, 100 and 200 g.mL-1) and incubation time of 48 h. In this part, MTT test was carried out under the same conditions with the above-mentioned experiments with the difference that before addition of MTT, the cell culture plate (including MCF-7, HeLa, Coav4 and HBL-100 cells and MNP-G2/MTX) was positioned under a permanent magnetic field to impose magnetic influence for 24 h, followed by another 24 h of incubation without any magnetic field. This is while in previous experiments, the cells were incubated for 48 h without the presence of the magnetic field. 2.4.4. Agglutination of RBCs. In vitro hemagglutination of human blood was performed to evaluate the hemagglutination activity of nanoparticles on RBCs. 80 µL of fresh human blood was exposed to 20 µL of G2 and G2-MTX at concentrations of 5, 10, 50, 100, 200 µg.mL-1, incubated at 37 °C for 30 min and then centrifuged at 1500 rpm for 5 min to separate plasma from RBCs. 20 µL of the supernatant as plasma was discarded and the same volume of fresh plasma replaced.


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For the studies of agglutination, 2 µl of resulting mixture (the mentioned suspension) and 20 µl of PBS were placed on a glass slide, covered with a coverslip and analyzed by a phase contrast microscope Olympus BX41, Olympus, Japan. 2.4.5. Coagulation Time. To perform PT and APTT tests, at the first fresh blood of healthy volunteers was mixed with sodium citrate. By the centrifugation (1500 rpm for 20 min) of the mixture, plasma was obtained as a yellow color supernatant. 450 µL of fresh human plasma was mixed with 50 µL of different concentrations (5, 10, 50, 100 and 200 µg.mL-1) of MNP-G2 and MNP-G2/MTX in PBS and incubating at 37 °C for 30 min. The sample of plasma mixing with PBS was set as control. To measure PT, PT reagent (Thromboplastin) was added to activate external clotting route and for APTT determination, APTT reagent and CaCl2 were added to initial the internal clotting cascade. Finally, the analysis of PT and APTT was carried out by a fully automatic blood coagulation analyzer (IL ACL Elite Pro, USA). 2.4.6. Complement Activation. Assessment of C3 and C4 complement components is an indicator of complement system activation. Briefly, 100 µl of human serum was added to 100 µl of nanoparticle samples with 5 different concentrations (5, 10, 50, 100, 200 µg.mL-1) and the mixture was incubated at 37 °C for 30 min. A mixture of human serum and PBS was applied to the control sample. Subsequently, the ready-to-use reagents were added to the mixture and activation of C3 and C4 were quantified by using a Roche/Hitachi 902 auto analyzer. 2.4.7. Preparation of Fluorescently Labeled MNP-G2/MTX (FMNP-G2/MTX). The resulting nanocarrier was fluorescently labeled with FITC. This means that a methanolic solution of FITC was added slowly to the appropriate quantities of MNP-G2 in phosphate-buffered saline (PBS; pH 7.4). (dendrimer: FITC weight ratio 15:1). The reaction was carried out for 24 h at room temperature and incubated in the dark with shaking. In the following, FMNP-G2/MTX was


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recovered from unreacted FITC by using magnetic separation and thoroughly washed with DI water. 2.4.8. Confocal Laser Scanning Microscopy. Confluent MCF-7 cell line at the density of 4×105 cells/well was cultivated on coverslips in 35 mm culture dishes as described above. Then, cells were treated with MNP-G2-FITC conjugate at various concentrations (2, 5, 10 and 30 µg.mL-1) at the conditions described earlier. After 12 h, cells were washed with PBS and fixed with 4.0% formaldehyde for 5 min at room temperature. Nuclear visualization of cells was accomplished by 4'-6-diamidino-2-phenylindole (DAPI) which inserts into DNA and acts as cell nuclei marker. Cells were then washed three times with PBS and stained with DAPI/PBS solution for 5 min. Finally, the cells examined under a confocal laser scanning microscope (Nikon Ti microscope). The fluorescence excitation and emission wavelengths were 364 and 461 nm for DAPI and 488 and 518 nm for FITC, respectively. Investigation on the different fluorophores was performed through appropriate filters to assemble fluorescence signals. The imaging software in the confocal system (NIS elements) was used to detect co-localization. 2.4.9. Flow Cytomtry Assay. MCF-7 cells were seeded at 2×105 cells/ml in 6-well plates using RPMI 1640 cell culture medium supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin and incubated at 37 °C in an atmosphere of 5% CO2. When the cells were 80% confluent, FMNP-G2/MTX was added in different concentration and cells incubated for 6 h and 12 h. The cells were treated with trypsin, washed with phosphate buffered saline (PBS; pH 7.4) via centrifugation at 1500 rpm for 5 min. The pellet was then resuspended in PBS buffer, and intracellular fluorescence analyzed immediately using flow cytometry (FACS caliber, Becton Dickinson) based on the collected 10,000 cells and an excitation wavelength of 488 nm (emission wavelength 518 nm) was employed.


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2.4.10. Degradation of the MNP-G2. For each test, initially, 20 mg of MNP-G2 was dispersed in 10 mL of PBS at pH 7.4. Next, 1 mL of Lysozyme solution (5% w) was added to the suspension. The biodegradation process of MNP-G2 was carried out at 37 °C at different times. Then, the nanoparticles were removed from the reaction medium with a conventional permanent magnet and washed with DI water. After vacuum drying in 60 °C, the remained nanoparticles were weighted. 3. RESULTS AND DISCUSSION This section is divided into two total parts. The first part is about the design and synthesis of MNP-G2 and their physicochemical characterization, and the second one includes some in vitro experiments such as loading of nanoparticles with anticancer drug MTX, release profile of drug from nanocarrier, verifying of biocompatibility of MNP-G2/MTX via significant biological assessment.

3.1. Synthesis and Characterization of MNPs. The synthetic route of MNP-G2/MTX is shown in Scheme 1. The Fe3O4 nanoparticles were synthesized by controlled co-precipitation of FeCl2 and FeCl3 under excessive ammonia solution at room temperature. To improve the chemical stability of Fe3O4, coating of the surface of Fe3O4 nanoparticles with a layer of silica was achieved by the suitable deposition of SiO2 onto Fe3O4 surface by the ammonia-catalyzed hydrolysis of tetraethylorthosilicate (TEOS) via sol-gel process based on the modified Stöber method.53 In the next step, silica-coated Fe3O4 nanoparticles were amine activated by 3aminopropyltriethoxysilane (APTES). There are many silanol groups (Si−OH) on the surface of silica and these groups can be easily coupled with APTES through the grafting of aminopropyl silane groups via formation of covalent bonds (Si−O−Si). Finally, triazine dendrimer was grafted


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on the surface of aminopropyl-functionalized Fe3O4@SiO2 magnetic nanoparticles (MNP-AP) by using cyanuric chloride (CC) as the core reagent with special properties. The reaction of CC with the surface-attached propylamine of MNP-AP was carried out at 0 °C for substitution of one of the chlorine atoms to afford MNP-CC1. Then, the above-mentioned product was reacted with bis(3-aminopropyl)amine to give MNP-G1 (the first generation of triazine dendrimer supported on MNPs) which in turn was converted to MNP-CC2 upon reaction with cyanuric chloride. Finally, the MNP-CC2 reacted with bis(3-aminopropyl)amine which produced the second generation of triazine dendrimer supported on MNPs (MNP-G2) as a potential platform for drug loading. Scheme 1. Synthesis of Triazine Dendrimer Supported on Magnetic Nanoparticles and

2

Si

Si

2

NH

Encapsulation of MTX.

NH

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The structure and composition of synthesized nanoparticles were characterized by FT-IR, TGA, VSM, FE-SEM , TEM and DLS analyses. Fe3O4 was identified by the stretching vibration of the Fe−O absorption peak at 590 cm-1 and OH stretching vibration at 3435 cm-1 (Figure 1a). The FT-IR spectrum of Fe3O4@SiO2 (Figure 1b) displayed characteristic peaks at 1089 and 806 cm-1 corresponding to symmetric and asymmetric linear vibrations of Si−O−Si, respectively, confirming the presence of SiO2 layer around Fe3O4 core. The successful synthesis of MNP-CC1, MNP-G1, and MNP-G2 was proved by the appearance of the new bands at 1563-1608 cm-1 originated from the absorption of C=N bond and also the weak bands in 2930-2865 and 1408-1386 cm-1 related to the aliphatic C−H stretching and bending vibrations, respectively. Unfortunately, the stretching absorption of N−H bond was masked by O−H strong peak on the surface of SiO2 in 3200-3400 cm-1 (Figures 1c-e).

Figure 1. The FT-IR spectra of: (a) Fe3O4, (b) MNP (Fe3O4@SiO2), (c) MNP-CC1, (d) MNPG1, and (e) MNP-G2.


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The thermal stability and weight loss of the MNPs, MNP-CC1, MNP-G1, MNP -CC2, and MNPG2 was evaluated by TGA. According to the TGA curve (Supporting Information, Figure S1a), about 3% weight loss was observed for MNP below 800 °C. The TGA plots of MNP-CC1, MNP-G1, MNP-CC2 and MNP-G2 (Supporting Information, Figures S1b-e) depict a two-step thermal decomposition. The first step of weight loss in the case of the above-mentioned samples (between 30 to 200 °C) corresponds to the evaporation of physically adsorbed water and other solvents, whereas, the second weight loss step from 200°C to 800 °C is assigned to the thermal decomposition of organic moieties on the MNPs surface. The observed total weight loss for MNP-CC1, MNP-G1, MNP-CC2 and MNP-G2 are 5.23%, 9.12%, 19.41% and 29.86%, respectively. Base on obtained values, the theoretical conversion is 92.3% for MNPCC1→MNP-G1, 85.5% for MNP-G1→MNP-CC2, 81.4% for MNP-CC2→MNP-G2. The magnetic properties of Fe3O4 nanoparticles and MNP-G2 triazine nanocarrier were analyzed by VSM (Supporting Information, Figure S2). The amount of saturation magnetization for Fe3O4 and MNP-G2 were found to be 60.73 emu/g and 34.03 emu/g, respectively. The obvious decline in magnetic response implied an increase of the thickness of shell layer on the surface of Fe3O4 core during the modification procedure. Accordingly, the immobilization of SiO2modified G2 on Fe3O4 surface caused in the reduction of its magnetization properties. In addition, the magnetization curve exhibits zero remanence and coercivity, which proves that both uncoated Fe3O4 nanoparticles and MNP-G2 nanocarrier exhibit typical superparamagnetic behavior. Thus, the MNP-G2 is able to respond to an external magnetic field. The high-magnification FE-SEM image of nanocarrier platform (MNP-G2) in Figure 2a showed the surface morphology of nanoparticles with a nearly spherical shape. This image clearly displayed the particles diameter in the range of nanometers. So, the small dimensions of


Langmuir

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nanoparticles and large specific surface to volume ratio can lead to higher efficiency in the drug loading.

Figure 2. (a) FE-SEM and (b and c)TEM images of the MNP-G2. Further characterization of the magnetic nanocarrier was performed by TEM. As demonstrated in Figure 2b and 2c, MNP-G2 has the spherical morphology. In this Figure, two regions with different electron densities can be distinguished that confirms the Fe3O4 nanoparticles have been successfully encapsulated in a shell of SiO2-modified G2 triazine with a different phase. The dark core in the electron-dense region is related to Fe3O4 nanoparticles, and the light shell in less dense or more translucent region surrounding these cores corresponds to SiO2-modified G2 shell. However, it can be observed that the sample is nearly in core-shell structure. The zeta potential of MNP-G2 was measured at three different pH values (7.4, 6.5 and 5.5). As can be seen in Figure 3a, the zeta potentials of MNP-G2 changed from negative value at pH 7.4 to positive value at pH 5.4, due to the enhanced protonation of the amino groups of triazine dendrimer segments. Moreover, the low value in neutral medium suggested that the dispersed nanoparticles were stable mostly, because of steric repulsion of branches of dendrimer structure. The branches of the MNP-G2 associated with water molecules, preventing agglomeration.


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Langmuir

Remarkably, the particle sizes of the MNP-G2 remains essentially constant at pH 5.5, 6.0 and 7.4, as partly represented in Figure 3b. This property is exclusively important that the MNP-G2 which shows positive surface, maintained a particle size of ca. 46 nm when exposed to the week acidic environment of cancer cells, a vital prerequisite for the permeation into deep tumor tissue in vivo.56, 57

Figure 3. (a) Zeta potential and (b) DLS particle size distribution profiles of MNP-G2 in aqueous solutions. Thus, MNP-G2 triazine platform was identified by using the techniques described above and applied for loading of MTX. 3.2. In vitro Studies 3.2.1. MTX Loading and Release Profiles. MTX loading amount on MNP and MNP-G2 was achieved by adding different concentrations of MTX solutions into an aqueous dispersed solution of MNP and MNP-G2 as nanocarries with a constant concentration (0.13 mg.mL−1). Then, the loading capacity was determined by UV spectra based on the difference of MTX concentrations between the original MTX solution and the supernatant solution after loading. As can be seen in Figure 4a, the loading of MTX on MNP and MNP-G2 were 0.06 and 0.32 mg.mg-1 at the MTX


Langmuir

concentration of 0.07 mg.mL-1, respectively. After increasing of the MTX concentration, the loading capacity of MTX on MNP-G2 enhanced linearly and reached to 0.66 mg.mg-1 at the MTX concentration of 0.115 mg.mL-1, while the significant changes were not observed for loading of MTX on MNP as simple carrier. This observation shows that high loading of MTX on MNP-G2 can be attributed to elegant three dimensional structure of triazine dendrimer with extremely high cavities and branches. Besides, the hydrophobic effects and hydrogen bonding between triazine dendrimer and MTX result in greater retention of the drug inside cavities of dendrimer and consequently, the high capacity of MNP-G2 carrier for acceptance of the MTX.

0.35

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Loading MTX on MNP-G2 (mg/mg)

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Loading M TX on M N P-G2 (mg/mg)

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MNP-G2 MNP

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Figure 4. The loading capacity of MTX on MNP and MNP-G2 in different initial MTX concentrations (a) and at different pH values (b). Additionally, the loading potential of dendrimer nanocarriers is dependent on the type of drug and loading conditions.58, 59 MTX is an acidic hydrophobic anti-cancer drug with bulky pteridine ring as well as massive constituents.60 Moreover, the triazine dendritic structure contains of amino groups and hydrophobic ducts that act as suitable host for diverse biological and drug molecules. Hence, the optimization of MTX Loading was first carried out as a function of pH


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Langmuir

and the loading efficiency was detected to be significantly high at pH 7.4 compare to acidic and alkaline pH conditions (Figure 4b). At this pH (pH 7.4), the pKa of MTX stands on 4.8 and accordingly, the dendrimer remains greatly ionized at pH 7.4 compared to acidic pH. Also, simultaneously peripheral and tertiary amines of the dendrimer are protonated around neutral pH. On the other hand, the repulsion between protonated amine groups creates large and more configured internal cargo spaces for MTX loading. As a whole, this panorama causes the increasing the loading of MTX (including hydrophobic and electrostatic interactions) and it can be one of the most probable reasons for enhancing drug loading observed at pH 7.4. It is well known that at low pH, the dendrimer is protonated also the crevices development are increased. However, at the same time it should also be noted that by increasing of internal voids space, the dendrimer architecture becomes so open that the loaded drug can’t be retained there for a long time and frequently leaks out. The lowest drug loading occurred at pH 3.5, may be due to the complete protonation of the dendrimer scaffold and non-ionized nature of MTX (pH below pKa). Also at alkaline pH (8.5 and 9.5), despite the fact that the degree of dendrimer deprotonation is sufficiently high, however, the MTX is ionized and stays onto nano carrier via hydrogen bonding with primary amines, and the drug loading under this pH is not satisfactory. The release profile for MNP-G2/MTX in phosphate-buffered saline (PBS) with pH 5.5 (the cancer cells endosomal pH) and 7.4 (the physiological pH) was investigated to evaluate how pH variation affected the release of drug from nanocarrier. Figure 5 shows the release amount of MTX as a function of time under different pH conditions. The release studies were continued up to 45 h. According to Figure 5, the percentage of the released MTX was about 25% within 45 h, showing that the MNP-G2 loaded with MTX was relatively stable at pH 7.4. On the contrary after 45 h, the totally released MTX reached about 70% at pH 5.5 demonstrating that the MNP-


Langmuir

G2/MTX was more stable at pH 7.4. Clearly, the remove of interactions between drug and carrier became far easier in the slightly acidic environment. In total, MTX release was higher at pH 5.5 compared with pH 7.4.

80 70

Released MTX (%)

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60 50

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Figure 5. In vitro MTX release from MNP-G2 at pH 7.4 and 5.5.

3.2.2. Hemolysis Assay. The hemocompatibility of the MNP-G2 and MNP-G2/MTX was studied by hemolysis experiments (Supporting Information, Table S1). RBCs can be used to visualize and quantify the membrane interactions of carriers by determining erythrocyte aggregation or hemolysis. However, the main source of hemolysis might be the effects that the drug moiety produces on the carrier surface. The hemolysis percentages of samples were calculated by comparing their absorbance with the positive and negative controls according to the equation below: 61


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= 100% ×

−

−

Table S1 (see Supporting Information) showed the concentration dependent hemolysis and perturbation of RBCs after incubation with MNP-G2 and MNP-G2/MTX. From the data summarized in Table S1, it can be observed that MNP-G2 and MNP-G2/MTX samples did not induce any undesirable response and were non-hemolytic (less than 5%) at all concentration ranges. The highest hemolysis percentage was obtained 0.67% related to MNP-G2/MTX at a dose of 200 µg.mL-1 which was much less than the normal hemolysis value. As known, the kind and number of surface groups in a dendrimer can greatly influence the physical and biological properties in the periphery of a dendrimer. Accordingly, the negative hemolytic activity of MNP-G2 and MNP-G2/MTX demonstrated that the free NH2 groups on the surface of two mentioned samples have very partial interactions with RBCs membrane. 3.2.3. Cytotoxicity Assay. Cell viability or cytotoxicity can routinely be measured by quantification of mitochondrial succinate dehydrogenase activity of metabolically active cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent through colorimetric measurement. In the other hand, the MTT assay is based on the reduction of tetrazolium salt to a soluble red-colored formazan by cellular mitochondrial dehydrogenase of metabolically active cells. The amount of formazan dye (produced via the activity of dehydrogenase) is directly correlated to the number of viable cells. The results were normalized to the control cells, and the cell viability was calculated using the following formula:62


Langmuir

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Cell viability (% of control) = [(Atest - Ablank/Acontrol - Ablank)] ×100%

In cell viability studies, the cytotoxicities of MNP-G2, free MTX, and MNP-G2/MTX were compared to further verify the efficacy of magnetic targeting. To this purpose, tumor cell lines including MCF-7 (breast cancer cells), HeLa cells (cervical cancer cells), Caov-4 (ovarian cancer cells) and HBL-100 (normal breast epithelial cells) were incubated for 24 h and 48 h in the concentration range from 5 to 200 µg.mL-1 of MNP-G2, free MTX, and MNP-G2/MTX. The results clearly proved that the activity of MTX-loaded on the dendrimer carrier against all above three mentioned tumor cells was greater than that of MNP-G2 and free MTX in all concentrations. The MTT assay in Figure 6a exhibited the moderate cytotoxic effects of free MTX and MNPG2/MTX (>57% of viability) on MCF-7 cells after 24 h incubation in different concentrations, while after an incubation time of 48 h, the viability of MCF-7 cells reduced especially in the presence of MNP-G2/MTX. According to Figure 6a, the percentage of MCF-7 cells viability in MNP-G2/MTX with concentration of 200 µg.mL-1 is 45.1% that verifies higher antitumor effects of the mentioned nanoparticle against 52.02% of viability in free MTX with concentration of 200 µg.mL-1. As shown in Figure 6b, the cytotoxicity effect of the MNP-G2/MTX against HBL-100 normal breast cell line did not exhibit significant cytotoxicity at lower concentration in 24 h. But, the cytotoxicity increases with increasing concentration of the MNP-G2/MTX by 200 mg.mL-1 in 24 h and 48 h. These results provide conclusive and logical evidence for cytotoxic effect of the MNP-G2/MTX against cancer cell lines compared with normal cells.


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Langmuir

Figure 6. (a, c and d) The effect of different concentrations of free MTX, MNP-G2/MTX, and MNP-G2/MTX + MF and MTT viability assay on the survival rates of MCF-7, HeLa and Coav4 cells, respectively, and (b) The effect of different concentrations of MNP-G2/MTX on HBL100 after 24 and 48 hours. Data are expressed as the mean of three independent experiments. Statistical analyses were performed using ANOVA followed by Tukey’s multiple comparison tests. Values are mean ±SD, *p

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