Methotrexate-conjugated chitosan-grafted pH- and thermo-responsive magnetic nanoparticles for targeted therapy of ovarian cancer

Journal Pre-proofs Methotrexate-conjugated chitosan-grafted pH- and thermo-responsive magnetic nanoparticles for targeted therapy of ovarian cancer Marziyeh Fathi, Jaleh Barar, Hamid Erfan-Niya, Yadollah Omidi PII: DOI: Reference:

S0141-8130(19)35951-3 https://doi.org/10.1016/j.ijbiomac.2019.10.272 BIOMAC 13773

To appear in:

International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

29 July 2019 8 October 2019 30 October 2019

Please cite this article as: M. Fathi, J. Barar, H. Erfan-Niya, Y. Omidi, Methotrexate-conjugated chitosan-grafted pH- and thermo-responsive magnetic nanoparticles for targeted therapy of ovarian cancer, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.272

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Methotrexate-conjugated chitosan-grafted pH- and thermo-responsive magnetic nanoparticles for targeted therapy of ovarian cancer Marziyeh Fathi1, Jaleh Barar1,2, Hamid Erfan-Niya3, Yadollah Omidi1,2,*

1

Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical

Sciences, Tabriz, Iran 2

Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

3

Faculty of Chemical and Petroleum Engineering, University of Tabriz, Tabriz, Iran

*

Corresponding author: Yadollah Omidi [email protected]

1

Abstract Here, methotrexate (MTX)-conjugated multifunctional nanoparticles (NPs) were engineered based on chitosan (CS) for target delivery of erlotinib (ETB). First, CS was modified with sodium dodecyl sulfate and maleic anhydride to prepare a polymerizable organo-soluble precursor. N-Isopropylacrylamide (NIPAAm) and itaconic acid (IA) as thermo- and pHresponsive monomers were grafted onto CS by free radical polymerization. Subsequently, magnetic nanoparticles (MNPs) ferrofluid with prepared CS copolymer (CSC) was produced via an inclusion complex between carboxylic acid groups of CSC and MNPs. Finally, MTX was conjugated to amino groups of CS based on the structural similarity of MTX with folic acid as targeting ligand. The prepared MTX-CSC@MNPs were physiochemically characterized by different techniques, including FT-IR, 1H NMR, DLS, SEM-EDX, and UVVis spectroscopy. The mean size of the prepared NPs was around 112 nm with a zeta potential of -28.4 mV. ETB encapsulation efficiency and NPs loading were about 86 % and 17 %, respectively. The ETB loaded NPs showed thermo- and pH-dependent drug release behavior. The ETB-loaded MTX-CSC@MNPs were markedly taken up via folate receptors (FRs) and induced significant toxicity in OVCAR-3 cells. Based on these findings, MTXCSC@MNPs are proposed as a promising smart nanocarrier for the targeted therapy of FRpositive solid tumors.

Keywords: Chitosan; Cancer therapy; Erlotinib; pH-responsive; Thermo-sensitive; Targeted drug delivery

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1- Introduction Despite remarkable advancements in cancer treatment using chemotherapeutics, still, there exist striking challenges upon the specific discrimination of cancer cells from healthy normal cells and the effective eradication of the diseased cells [1, 2]. To tackle such shortcomings, nanoscaled targeted delivery of anticancer agents with stimuli-responsive potential have been engineered to guarantee the efficient delivery of anticancer chemotherapeutics selectively to the diseased cells/tissues through passive and/or active targeting mechanisms, i.e., enhanced permeability and retention (EPR) effect and decoration of nanoparticles (NPs) with homing agents [3-7]. Once delivered into the tumor microenvironment (TME) and attached to the diseased cells, the release of cargo drug molecules can be triggered in response to the stimuli [8-10]. To date, different types of nanoscaled drug delivery systems (DDSs) have been developed for the delivery of anticancer agents, while the ideal DDSs are those that provide simultaneous diagnosis and treatment options. In this line, multifunctional magnetic nanoparticles (MNPs) are deemed to offer such potentials, for which an external magnetic field can be used to concentrate the MNPs around solid tumors [11-14]. MNPs are considered as less toxic inorganic NPs, which can be further coated with various materials (e.g., natural/synthetic polymers, peptides, fibronectin, PEG, and targeting agents) to improve their biocompatibility and pharmacokinetics (PK) properties [15]. Surface modified targeted MNPs loaded with anticancer drugs can reach the TME and be endocytosed by cancerous cells. Next, in the endosomal vesicles of cancer cells, the biodegradable linkage of the NPs is cleaved (by enzymes, pH or thermal changes) and the cargo drug molecules are released into the target cells [16-19]. In fact, modification of MNPs with stimuli-responsive polymeric biomaterials could be performed using internal and/or external stimuli [20-22]. In this line, carboxylic functional groups of polymers can electrostatically interact with MNPs and make a stable

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MNPs fluid [20]. Due to good biocompatibility and biodegradability, natural polymers have extensively been used in biomedical applications [23]. Among them, chitosan (CS), as a biocompatible and biodegradable polymer with abundant functional groups, holds great potential for the fabrication of advanced seamless DDSs with multimodal functions for simultaneous targeting, imaging, and therapy [24-28]. Coating MNPs with CS modify the surface properties of the core structure and improve their stability and biodegradation in surrounding media [29]. CS is a slowly biodegrading polymer, whose modification with other molecules with appropriate functional groups (e.g., carboxylic, ammonium and amides) may improve its biodegradation feature attributable to the initial crystalline structure destruction [30]. Furthermore, dual thermo/pH-responsive DDSs can serve on-demand drug release in response to an internal and/or external stimuli [23]. Of these, a pH-sensitive DDS has widely been used for the efficient delivery of chemotherapeutics due to the lower pH of the endosomal compartments of tumor cells as well as the tumor interstitial fluid [31]. Poly(N-isopropylacrylamide) (PNIPAAm) is the most studied thermo-responsive polymer which exhibits a lower critical solution temperature (LCST) in the water around 32°C. PNIPAAm-based DDSs can release the entrapped drug molecules in response to the changes of temperature in a controlled manner. Graft copolymerization of N-isopropylacrylamide (NIPAAm) with CS could result in the production of thermo-sensitive biocompatible drug delivery carrier [32, 33]. Further, the incorporation of hydrophilic moieties such as itaconic acid (IA) into the PNIPAAm structure can increase the phase transition temperature nearly up to the body temperature, which makes it a pH-responsive entity with an enhanced drug release in acidic environments [4, 34]. Methotrexate (MTX) is one of the most widely used chemotherapeutics for the treatment of a wide range of cancers. Structurally, MTX is similar to folic acid (FA) and can target the

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folate receptors (FRs) along with its chemotherapy impacts in the FR-positive cancers [3538]. The poor solubility of CS restricts its widespread applications, and therefore, various modification of CS has been carried out to increase its water solubility for various biomedical purposes [39]. Here, we have engineered a novel CS-based thermo/pH-sensitive multifunctional nanosystem (NS) containing a magnetic core as a theranostic agent for the delivery of tyrosine kinase inhibitor, erlotinib (ETB). Further, MTX was conjugated to the surface of nanoparticles (NPs) for active targeting of FR-positive ovarian cancer cells. First, CS was modified to become an organo-soluble, polymerizable and amino protected precursor. Second, thermo- and pH-responsive water-soluble copolymer was synthesized via free radical polymerization of NIPAAm and itaconic acid (IA) on CS. Third, CS copolymer coated MNPs (CSC@MNPs) was obtained by forming an inclusion complex between carboxylic acid groups of CS copolymer and hydroxyl groups of Fe3O4 NPs. Finally, MTX was covalently conjugated onto the amino group of CS as a targeting agent (MTX CSC@MNPs), and then, ETB molecules were physically loaded on the MTX-CSC@MNPs. The physicochemical properties, the cellular uptake, and the cytotoxicity of the engineered NS were investigated.

2- Method and materials 2.1. Materials Chitosan (medium molecular weight with N-deacetylation degree of (75–85%)), dialysis membranes (benzoylated dialysis tubing, MWCO = 2000 and 12000 Da), maleic anhydride, fluorescein isothiocyanate (FITC), sodium dodecyl sulfate (SDS), N, N-dimethylformamide (anhydrous) (DMF), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) were obtained from Sigma-Aldrich Corp. (St.

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Louis, MO, USA). NIPAAm (99%) and azobisisobutyronitrile (AIBN) were obtained from ExirPharma Co. (Boroujerd City, Iran). Acetic acid (glacial), itaconic acid (IA), dimethyl sulfoxide (DMSO), ferrous chloride tetrahydrate (FeCl2 4H2O) and ferric chloride hexahydrate (FeCl3 6H2O) were purchased from Merck (Kenilworth, NJ, USA). Human ovarian carcinoma OVCAR-3 cell line was obtained from the National Cell Bank of Iran, Pasteur Institute (Tehran, Iran). All media and cell culture components were purchased from Invitrogen (Karlsruhe, Germany).

2.2. Instrumentation 2.2.1. Fourier transform infrared (FT-IR) FT-IR analysis was carried out to validate the synthesized materials. The FT-IR spectra were obtained using KBr disks on FT-IR Tensor 27 spectrometer (Bruker Optik GmbH, Ettlingen, Germany) in the range of 4000–400 cm−1.

2.2.2. 1H Nuclear magnetic resonance (NMR) 1

H NMR spectrum of CS copolymer was obtained on a 400 MHz NMR spectrometer (Bruker

Optik GmbH, Ettlingen, Germany) at 25°C in D2O.

2.2.3. Dynamic light scattering (DLS) analysis DLS was used to determine the particle size distribution, polydispersity index (PDI) and zeta potential of the prepared MTX-CSC@MNPs in aqueous suspension with a concentration of 1 mg/mL using Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). All analyses were performed at room temperature.

2.2.4. Morphologic characterization

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The surface morphology, size, and element analysis of MTX-CSC@MNPs were determined by means of TESCAN MIRA3 electron microscope (TESCAN, Brno, Czech) equipped with energy dispersive X-ray (EDX) and conducted as file emission scanning electron microscopy (FE-SEM-EDX). The samples were coated with gold prior to imaging. The contents of different elements including C, O, N, and Fe in the synthesized NPs were estimated as the percentage of their related peaks by area in the EDX spectra.

2.3. Synthesis of chitosan copolymer (CSC) Maleic anhydride modified CS as a polymerizable precursor, with regioselective conjugation potent via protected amino groups, was synthesized according to our previous published work [40]. Subsequently, graft copolymerization of NIPAAm/IA to the modified CS was carried out in dried DMF using AIBN as the initiator via free radical polymerization under a nitrogen atmosphere. Briefly, modified CS (1 g) was dissolved in DMF (30 mL). Then, NIPAAm (1 g, 0.09 mol) and IA (120 mg, 0.92 mmol) were added and stirred under a nitrogen atmosphere at room temperature for 30 min. Afterward, the reaction temperature was raised to 75°C and AIBN (32 mg, 0.2 mmol in 1 mL DMF) was injected into the reaction system to initiate the polymerization process that was performed at 75°C under a nitrogen atmosphere with continuous stirring for 24 h. After cooling to room temperature, to remove SDS complex and unreacted moieties, the mixture was dialyzed against 15% Tris aqueous solution (pH = 8.0) with cellulose membrane (molecular cut-off 12000 Da) for 24 h, and then, against water/ethanol mixture for 48 h. Finally, the solution was freeze-dried. For further purification, the yielded product was dissolved in tetrahydrofuran (THF) and precipitated with excess cooled diethyl ether and dried in a vacuum oven.

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2.4. Synthesis of magnetic iron oxide nanoparticles (Fe3O4 MNPs) Fe3O4 MNPs were synthesized without the additional stabilizer using a chemical coprecipitation method [41]. Briefly, about 5.40 g of FeCl3.6H2O and 2 g of FeCl2.4H2O (with a mole ratio of 2:1) was dissolved in 50 mL deionized water. About 100 mL of 2 M NaOH solution was placed in a three-necked flask and the solution of FeCl3/FeCl2 was added dropwise to the NaOH solution. The mixture was vigorously stirred under N2 atmosphere for 2 h. The obtained MNPs were washed several times with first deionized water and then ethanol. The obtained product was dried in a vacuum oven at 50°C for 12 h and stored under vacuum.

2.5. Synthesis of chitosan copolymer-MNPs ferrofluid (CSC@MNPs) In order to CSC@MNPs preparation, about 100 mg of CSC was dissolved in 10 mL deionized water and 100 mg of MNPs was added to the copolymer solutions, and then, vigorously stirred at room temperature for 24 h. To separate the obtained magnetic ferrofluid from any unmodified Fe3O4 particles and undispersed residue, the mixture was centrifuged at 6000 rpm for 10 min. Finally, to remove the excess copolymer, the obtained ferrofluid was enriched by means of an external magnet and washed (×3) with distilled water and then dried in a vacuum oven.

2.6. Synthesis of MTX conjugated nanoparticles MTX (5 mg, 0.01 mmol) was activated with EDC (3 mg, 0.02 mmol) and NHS (5 mg, 0.044 mmol) in 6 mL dried CH2Cl2 and then stirred at room temperature for 12 h. Subsequently, 100 mg CSC@MNPs was dispersed in 5 mL dried DMSO and added to the activated MTX reaction system and stirred vigorously for 24 h. Next, the MTX-conjugated product was separated by an external magnet and washed with ethanol (×3) and then dried in a vacuum

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oven at 50°C for 4 h. The conjugation of MTX was confirmed by means of UV-Vis spectrophotometer at 387 nm.

2.7. Erlotinib loading and in vitro release analysis For the loading of ETB, 4 mg of ETB was added to a 20 mg/mL well-dispersed suspension of MTX-CSC@MNPs in distilled water and vigorously stirred at room temperature for 24 h. The ETB-loaded MTX-CSC@MNPs were literally collected by an external magnet and the supernatant was separated for the calculation of unloaded ETB by means of UV-Vis spectrophotometer at 333 nm (using calibration plot of ETB in PBS). To investigate the drug release, ETB-loaded NPs were dispersed in 5 mL PBS (6 mg/mL) and placed into a dialysis bag (molecular cut-off, 2.0 kDa). The dialysis bag was immersed in 30 mL of phosphate buffer (pH=7.4 and 5.5) at 37°C and 40°C to evaluate the temperature- and pH-dependent release of the loaded drug from NPs. To measure the amounts of the released drug, 2 mL of release medium was taken out from the solution at predetermined times and the same amount of fresh buffer was added after each sampling. The amount of cumulative released drug was calculated using UV absorbance.

2.8. In vitro cytotoxicity evaluation The cytotoxicity of CSC@MNPs, MTX-CSC@MNPs, ETB-loaded MTX-CSC@MNPs, and ETB alone was investigated in the human ovarian cancer OVCAR-3 cells. Briefly, cells were cultured at a seeding density of 5.0 × 103 cells/well in a 96-well plate using RPMI media and incubated (5% CO2) at 37 °C for 24 h. At 40-50 % confluency, the medium of cultivated cells was replaced with 200 µL fresh growth medium containing various concentrations of samples. The cells were further incubated for 24, 48 and 72 h. At designated time points, MTT solution (5 mg/mL) was added to each well. After the cells were incubated for further 4 9

h, the culture medium was removed. Then, the formed formazan crystals were dissolved in 200 µL DMSO. Finally, the optical density was recorded at 570 nm using a microplate reader, Elx808 (BioTek Instruments, Winooski, VT, USA) in comparison with the untreated control cells. 2.9. Cellular uptake assay The cellular uptake of the prepared NPs in the OVCAR-3 cells was determined using FITClabelled NPs by flow cytometry analysis. For the labeling of the NPs with FITC, the conjugation reaction was performed between amino groups of NPs and isothiocyanate according to a method described previously [42]. To this end, 100 µL of FITC solution in ethanol with a concentration of 1 mg/mL was added dropwise to the NPs suspension (2 mg/mL). The suspension was stirred at room temperature for 24 h in dark. The FITC-labelled NPs were separated using a magnetic field from the excess amount of FITC. They were then washed with deionized water repeatedly and finally dried in a vacuum oven. The cells were cultivated at a seeding density of 3.0 × 105 cells/well in six-well plates and incubated for 24 h. Then, they were treated with CSC@MNPs, MTX-CSC@MNPs and ETBloaded MTX-CSC@MNPs (equivalent to 20 µM) for 3 h. Thereafter, the cells were trypsinized and washed with PBS and analyzed by means of FACS Calibur® flow cytometer (Becton Dickinson, San Jose, CA, USA) in a minimum number of 1.0 × 104 cells/event to determine the fluorescent intensity associated to the uptake FITC-labelled NPs by the cells.

2.10. Apoptosis assay by FITC-labelled annexin V To study the cell apoptosis, the OVCAR3 cells were seeded in a density of 5.0×105 cells/well in six-well plates in the culture medium. After 24 h, the cell culture mediums were removed and replaced with fresh medium containing ETB, MTX and ETB-loaded MTX-CSC@MNPs (equivalent to 20 µM) and the cells were further incubated for 48 h. Subsequently, the cells 10

were trypsinized, detached and collected. The cells were then incubated with 100 µL binding buffer and the apoptotic cells were stained via a FITC-annexin V apoptosis detection kit according to the manufacturer's protocol. Finally, FACS-Calibur flow cytometry (Becton Dickinson, San Jose, CA, USA) was used to detect the apoptotic cells. 2.11. Statistical analysis The statistical analyses were performed by either Student's t-test or ANOVA assay followed by a multiple comparison post hoc test. Data are represented as the mean values ± standard deviations (SD). A p-value of less than 0.05 was considered statistically significant.

3- Results and discussion

3.1. Preparation and characterization of MTX-CSC@MNPs

Due to the insolubility of CS in organic solvents, it was modified with SDS based on our previously published work [40, 43]. The SDS-modified CS was further functionalized with maleic anhydride (MA) for the polymerization of NIPAAm and IA to incorporate thermal and pH sensitivity properties, respectively (Fig. 1). The synthesis and characterization of SDS and MA modified CS was performed according to our previous work [40]. The solubility of modified CS with MA and CSC was significantly increased as compared to CS alone. The stable aqueous magnetic fluid was achieved via adsorption of carboxylic functional groups from both (MA) and IA moieties onto the surface of MNPs. MTX was anchored on the surface of CSC@MNPs via covalently imide bond. Scheme 1 indicates the synthesis steps of MTX-CSC@MNPs schematically. The FT-IR spectra confirmed the successful grafting of NIPAAm and IA on the modified-CS via free radical copolymerization (Fig. 2). The

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characteristics peaks of PNIPAAm, amid I and II, appeared at 1650 and 1541 cm-1, respectively. A shoulder peak related to the carbonyl bond of IA appeared around 1718 cm-1. The peaks corresponding to C-O stretching vibration of CS appeared around 1059 cm-1 [40]. The structure of CSC was further confirmed by the 1H NMR spectrum of CSC in D2O (Fig. 3). The protons of the main chain of NIPAAm and IA (CH2 and CH) appeared around 1.2-2 ppm. Peaks at 3.7 and 1.0 ppm could be assigned to protons of isopropyl group (–NHCH, CH3) of PNIPAAm [44]. Peaks at 2.7, 2.8 and 3.5–4.0 ppm could be attributed to CS moiety [43, 45].

Fig. 1. Synthesis steps of chitosan copolymer.

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Scheme 1. Schematic presentation of MTX-CSC@MNPs preparation.

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Fig. 2. FT-IR spectra of CSC and MTX-CSC@MNPs.

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Fig. 3. 1H NMR of CSC in D2O.

As CSC holds similar functional groups, many peaks that appeared in the FT-IR spectrum of MTX-CSC@MNPs (Fig. 2) can also be featured in CSC spectrum. The characteristic band for C=N stretching of MTX in the FT-IR spectrum appears at 1639 cm-1 [37, 46]. Some other characteristic peaks related to amine deformation, carboxylic stretch and C-H of phenyls group appeared around 1625 cm-1, 2949 cm-1, and 1201 cm-1, respectively. In the FT-IR spectrum of MTX-CSC@MNPs, a new peak related to Fe–O characteristic absorption band was observed at 592 cm-1 [45]. In addition, the UV-Vis spectrum confirmed the conjugation of MTX. For CSC, there was no significant absorption above 200 nm in the UV–Vis, while the spectra of MTX and MTX-CSC@MNPs show a strong absorption peak at 387 nm (Fig. 4) [46].

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Fig. 4. UV-Vis spectra of CSC, MTX, and MTX-CSC@MNPs.

3.2. DLS analysis Particle size and zeta potentials of NPs are regarded as important parameters that affect their stability and biodistribution [47]. Fig. 5 shows particle size distribution, PDI and zeta potential of MTX-CSC@MNPs. The average hydrodynamic diameter of NPs was in the range of 112 nm and the zeta potential was around -28.4 mV. Despite the positive charge of CS, the negative charge of synthesized particles is attributable to the modification of CS with moieties that contain acidic groups. In addition, some of the amines of CS were replaced with MTX, which also shifts the zeta potential toward negative values.

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Fig. 5. Dynamic light scattering analysis of MTX-CSC@MNPs. (a) The particle size. (b) The zeta potential.

3.3. Morphology and elemental composition of MTX-CSC@MNPs by FE-SEM-EDX

The morphology of MTX-CSC@MNPs was investigated by FE-SEM-EDX. The SEM image of MTX-CSC@MNPs (Fig. 6a) indicates spherical shape and smooth surface with distribution around 20-60 nm. The variation of the size of NPs by SEM and DLS analysis may be attributed to the hydrodynamic size of NPs in the aqueous dispersion state in DLS analysis, which might be larger than that of the SEM analysis. On the other hand in SEM analysis the surface of the MNPs is collapsed during the imaging process in the dry state, resulting in a decreased size [29].

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The elemental composition and presence of C, N, O and Fe elements of MTX-CSC@MNPs were qualitatively analyzed by energy-dispersive X-ray (EDX). Based on the EDX spectrum (Fig. 6b), the presence of C, N, O, and Fe was confirmed with the percent weight of 32.13, 8.95, 56.62 and 23.56 %, respectively.

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Fig. 6. Surface morphology and composition of MTX-CSC@MNPs. (a) SEM micrograph. (b) EDX spectrum.

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3.4. ETB loading and release evaluation Erlotinib hydrochloride was loaded on the MTX-CSC@MNPs simply via vigorous stirring in PBS and unloaded drug was removed using an external magnet and washing (×3) with PBS, which were then subjected to the encapsulation efficiency (EE) and drug loading (DL) analyses. The EE and DL were found to be about 86 % and 17 %, respectively. The pH and temperature-sensitive characteristics of engineered NPs can enhance the chance of drug release in the target site. To investigate the pH- and temperature-dependent release behavior of formulation, the cumulative release of drug was evaluated as a function of time in simulated cancerous condition (i.e., 40°C, pH=5.5) and physiologic condition at 37°C and pH=7.4 (Fig. 7). Having considering drug release profiles at different pH and temperatures for up to 5 days, ETB molecules released more rapidly at pH 5.5 as compared to that of pH 7.4. This may be due to the protonation of NH2 functional groups of CS chain, which might increase the swelling of NPs and promote the drug release. Further, carboxylic groups could modulate the phase transition at lower pH and accelerate the release of loaded drug molecules [4, 32]. In a thermo-sensitive DDS, the temperature-dependent swelling/deswelling process controls the diffusion of the drug from NPs via steric interactions between the drug and polymeric entities. Since the steric and hydrophobic interactions increase at 40˚C compared to 37˚C, the drug release rate might decrease [5, 29]. On the other hand, pH-responsive DDS results in efficient drug release in the acidic tumor microenvironment, resulting in minimized drug release and hence cytotoxicity in normal physiological pH of the healthy tissue [48]. Various kinetic models were examined to fit the ETB release profile at different temperatures and pHs (Table 1) [49], with results showing that the ETB release profiles were fit best with the Korsemeyer-Peppas model (the so-called power-law). Based on the value of n, this kinetic model is often used to describe the Fickian/no-Fickian release profile of various complex phenomena, including (i) swelling of polymeric matrix through which water can 20

enter, (ii) formation of the gel, (iii) diffusion of drug and filler out of the polymeric matrix, (iv) overall dissolution of the polymer matrix. Upon these depictions, therefore, CSC@MNPs used in our study might swell and form a gel-like structure through which the drug release happens in a complex manner [50-53].

Fig. 7. Temperature- and pH-dependent cumulative release of erlotinib from MTXCSC@MNPs. Inset: Drug release during the first 4 h period.

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Table 1 The kinetics models of the ETB release from MTX-CSC@MNPs at various temperatures and pHs. Kinetics model

Coefficient of determination (R2)

Equation

T=37°C, pH=5.5

T=37°C, pH=7.4

T=40°C, pH=5.5

T=40°C, pH=7.4

Zero order

F  k0t

0.6323

0.4279

0.7529

0.5834

First-order

ln(1  F )  k f t

0.7622

0.5046

0.8135

0.6298

Higuchi

F  kH t

0.7628

0.6033

0.8840

0.7580

Power law

ln F  ln kP  P ln t

0.9736

0.9450

0.9683

0.9584

Square root of mass

1  1  F  k1/2t

0.7004

0.4662

0.7847

0.6074

Hixson–Crowell

1  3 1  F  k1/3t

0.7218

0.4790

0.7947

0.6150

Three seconds’ root of mass

1  3 (1  F ) 2  k2/3t

0.6782

04534

0.7745

0.5996

Weibull

ln( ln(1  F ))   ln td   ln t

0.9693

0.9532

0.9625

0.9572

Reciprocal powered time

1  m   1  b F  t

0.9613

0.9596

0.9563

0.9555

3.5. In-vitro cytotoxicity and cellular uptake evaluation The cytotoxicity of various concentrations of the CSC@MNPs, MTX-CSC@MNPs, ETBloaded MTX-CSC@MNPs, and ETB alone was evaluated in the OVCAR-3 cells via MTT assay for different time points (24, 48, and 72 h). As shown in Fig. 8, there was no cytotoxicity of CSC@MNPs in the treated OVCAR-3 cells with the concentrations tested, which indicate the biocompatibility of the engineered NPs. Slightly higher viability of cells treated with the NPs after 48 h may attribute to the possible magnetic particles with the absorption of Formazen dye and possible cellular adaptation [54, 55]. MTX-CSC@MNPs revealed some cytotoxicity in comparison to CSC@MNPs, in large part due to the presence of MTX molecules. The cytotoxic effects of both ETB-loaded MTX-CSC@MNPs and free ETB (at the same concentration) were found to be significantly higher than CSC@MNPs and

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MTX-CSC@MNPs. Several key factors (e.g., concentration, size, shape and surface coating, degradation and accumulation rate, biological features of target cells) can influence the undesired toxicity of nanoscaled formulations such as MNPs [15, 56]. A very low amount of MNPs was used in this study to avoid any unwanted toxicity by the nanocarrier itself. We speculate that the conjugated MTX molecules rather function as a targeting agent that can interact with the FRs expressed by the OVCAR-3 cells, resulting in an enhanced internalization. Intriguingly, ETB alone showed higher toxicity, perhaps via passive diffusion since ETB is a lipophilic agent. In the long-term, however, cancer cells can generate resistance mechanisms against free cytotoxic drugs which appears to be unlikely for the case of ETB-loaded MTX-CSC@MNPs. As shown in Fig. 9, the cellular uptake studies confirmed the association of the MTX-CSC@MNPs was significantly higher than that of CSC@MNPs. This indicates that the decoration of the NPs with MTX (as an FR substrate) could increase the cellular uptake by the FR-expressing OVCAR-3 cells. Thus, it can be deduced that MTX could be considered as a suitable targeting moiety for conjugation of NPs [57]. In this study, the percentage of the cells which uptake the CSC@MPNs has been evaluated, however, another important criterion for any MNP-based theranostic system is the quantity of the magnetic core within each cell. For revalidation, other techniques such as magnetic measurements, UV-Vis spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) can be implemented [58].

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Fig. 8. The viability analysis of OVCAR-3 cells treated with different concentrations of CSC@MNPs, MTX-CSC@MNPs, erlotinib (ETB)-loaded NPs (ETB-MTX-CSC@MNPs) and ETB. Panels a, b and c represent cell viability data after 24, 48 and 72 h incubation, respectively.

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Fig. 9. Flow cytometry analysis of the uptake of FITC-labeled CSC@MNPs, MTXCSC@MNPs, and erlotinib-loaded MTX-CSC@MNPs by OVCAR-3 after 3 h incubation period.

3.4. Cell apoptosis study To study the emergence of apoptosis, the effects of ETB alone, MTX-CSC@MNPs, and ETB-loaded MTX-CSC@MNPs in the OVCAR-3 cells were evaluated after 48 h treatment by means of flow cytometry analysis using annexin V/PI. As demonstrated in Fig. 10, ETBloaded MTX-CSC@MNPs could significantly inhibit the growth of OVCAR-3 cells as compared to the untreated control cells, the cells treated with MTX-CSC@MNPs, and free ETB. These findings revalidate the MTT cytotoxicity results, indicating higher internalization and greater induction of apoptosis of ETB-loaded MTX-CSC@MNPs in the OVCAR-3 cells.

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Fig. 10. Apoptosis in OVCAR-3 cells upon treatment with NPs after 48 h. (a) Untreated cells. (b) MTX-CSC@MNPs treated cells. (c) Erlotinib treated cells. (d) Erlotinib-loaded MTXCSC@MNPs. Quadrants are defined as upper left, necrotic; upper right, necrotic/late apoptotic; lower right, early apoptotic and lower left, viable cells.

4. Conclusion In this study, we engineered MTX-CSC@MNPs nanosystem that can be used for the delivery of anticancer agents. The NS was loaded with ETB molecules (ETB-loaded MTXCSC@MNPs) whose impacts were studied on the FR-positive OVCAR-3 cells to validate the effectiveness of the developed targeted NS in cancer therapy. The engineered NS displayed spherical morphology with high loading efficiency for ETB molecules together with thermoand pH-responsiveness. The drug release profile showed an increased release of drug molecules at pH= 5.5 and 37°C. The cellular uptake validated the markedly higher internalization of MTX-conjugated NPs, indicating targeting impacts of MTX molecules in

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FR-positive cells. The MTT cytotoxicity assay demonstrated significantly greater cytotoxicity for the ETB-loaded MTX-CSC@MNPs compared to free drug, which was revalidated by the apoptosis assay accomplished via flow cytometry analysis. Taken all, the engineered MTXCSC@MNPs nanosystem is proposed for the targeted therapy of a wide range of solid tumors. Nevertheless, prior to any clinical applications, the in vivo efficiency of the proposed NS should be verified by performing the experiments in animal models of ovarian cancer in the combination with external hyperthermia.

Acknowledgments Authors would like to acknowledge the Research Center for Pharmaceutical Nanotechnology (RCPN) at Tabriz University of Medical Sciences and Department of Chemical and Petroleum Engineering at the University of Tabriz (Tabriz, Iran) for technical support.

Conflict of Interest The authors declare no conflict of interest.

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