Magnetic and pH-Responsive Magnetite/Chitosan (Core/Shell) Nanoparticles for Dual-Targeted Methotrexate Delivery in Cancer Therapy

Magnetic and pH-Responsive Magnetite/Chitosan (Core/Shell) Nanoparticles for Dual-Targeted Methotrexate Delivery in Cancer Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Magnetic and pH-Responsive Magnetite/Chitosan (Core/Shell) Nanoparticles for Dual-Targeted Methotrexate Delivery in Cancer Therapy Ana Medina-Moreno, Mazen M. El-Hammadi, Gema I. Martínez-Soler, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4328624/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Sep, 2024 Read the published version in Drug Delivery and Translational Research → Version 1 posted 5 You are reading this latest preprint version Abstract Methotrexate successful therapy encounters various challenges in chemotherapy, such as poor oral bioavailability, low specificity, side effects and the development of drug resistances. In this study, we propose a dual-targeted nanocarrier comprising magnetite/chitosan nanoparticles for an efficient Methotrexate delivery. The synthesis of the particles was confirmed through morphological analysis using electron microscopy and elemental mappings via energy dispersive X-ray spectroscopy. These nanoparticles exhibited a size of ≈ 270 nm, a zeta potential of ≈ 24 mV, and magnetic responsiveness, as demonstrated by hysteresis cycle analysis and visual observations under a magnetic field. In addition, these core/shell particles displayed high stability, as evidenced by size and surface electric charge measurements, during storage at both 4 ºC and 25 ºC for at least 30 days. Electrophoretic properties were examined in relation to pH and ionic strength, confirming the stability. The nanoparticles demonstrated a pH-responsive drug release as observed by a sustained Methotrexate release over the next 90 h under pH ≈ 7.4, while complete release occurred within 3 h under acidic conditions (pH ≈ 5.5). In the ex vivo biocompatibility assessment, the magnetite/chitosan particles showed excellent hemocompatibility and no cytotoxic effects on normal MCF-10A and cancer MCF-7 cells. Furthermore, the Methotrexate-loaded nanoparticles significantly enhanced the antitumor activity reducing the half-maximal inhibitory concentration by ≈ 2.7-fold less compared to the free chemotherapeutic. cancer targeted therapy chitosan core/shell nanoparticles magnetic-driven nanoparticles magnetite Methotrexate pH-responsive drug release. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Methotrexate (MTX), an analogue of folic acid, is a powerful chemotherapeutic agent and widely used for the treatment of human malignancies and autoimmune diseases [ 1 , 2 ]. Its mechanism of action involves disrupting cellular folate metabolism by inhibiting dihydrofolate reductase, an essential enzyme for cell division. Despite its clinical efficacy, MTX faces challenges, including low specificity, dose-dependent undesirable side effects, the development of resistances in cancer cells and a potential risk of developing cancer [ 1 , 3 – 5 ]. Furthermore, MTX is a weak dicarboxylic acid [molecular weight, M W : 54.44 g/mol (C 20 H 22 N 8 O 5 ); pKa values of 3.8, 4.8 and 5.6] that exhibit a poor, pH-dependent solubility (aqueous solubility ≈ 0.01 mg/mL at 20°C) and low permeability (C log P ≈ 0.53). As a result, it has a poor oral bioavailability of less than 40%, which impairs its therapeutic effect, and therefore it is classified in class IV of the Biopharmaceutical Classification System [ 3 , 6 , 7 ]. In addition, MTX is chemically unstable and, thus, undergoes rapid decomposition when exposed to light or subjected to extremes of pH or temperature. Consequently, it is often administered intravenously at high doses (≥ 500 mg/m 2 ) [ 8 ]. To overcome these challenges, nanoparticles (NPs) have emerged as promising carriers for tailored MTX delivery [ 9 ]. Nanotechnology has revolutionized cancer therapy by enabling targeted drug delivery, minimizing side effects and enhancing treatment efficacy [ 10 – 17 ]. Nanocarriers act as sophisticated vehicles, circulating through biological barriers to reach the specific cellular environments where their cargo can exert the most significant therapeutic effect [ 11 , 14 , 18 – 23 ]. Numerous research endeavors have delved into the encapsulation of MTX within NPs, with the primary objective of overcoming drug resistances in cancer cells and enhancing therapeutic outcomes. The intricately tailored encapsulation within NPs aims to provide sustained release profiles, ensuring a prolonged and controlled delivery of MTX. This sustained release approach plays a pivotal role in circumventing the development of resistances. Furthermore, nanoformulations contribute to improved bioavailability, enhancing the amount of MTX reaching its target within the body, thereby maximizing its therapeutic impact [ 8 ]. Numerous polymers have recently been suggested to formulate MTX-loaded NPs for cancer therapy [ 4 , 24 – 29 ]. Among these polymers, chitosan (CS), a naturally derived polysaccharide from chitin, offers a wealth of advantages that render it an ideal candidate for use in the design of nanoparticulate drug delivery systems. Known for its hydrophilicity, biocompatibility, low immunogenicity, biodegradability and mucoadhesive properties, CS has found widespread application in diverse medical formulations [ 30 – 32 ]. CS’s appeal in the medical field extends beyond its biological properties to include its ease of modification and versatility. The presence of reactive amino and hydroxyl groups allows for various chemical modifications, facilitating the tailoring of CS-based formulations to meet specific medical requirements [ 33 – 35 ]. Furthermore, the cationic nature of chitosan enables interactions with negatively charged biological surfaces, a feature strategically employed in NP development [ 36 – 38 ]. When employed as a coating polymer for NPs, chitosan not only imparts stability and controlled release but also introduces an additional layer of biocompatibility, offering a hydrophilic stealth coating that prevent opsonization and evade phagocytosis in mononuclear phagocyte system, which is crucial for navigating the blood circulation effectively [ 39 – 45 ]. Several MTX-loaded CS-based NPs have been designed to enhance controlled drug delivery to tumors [ 46 – 49 ]. These include actively targeted NPs, such as those using covalently conjugated to folate [ 48 ], as well as stimuli-responsive particles. Stimuli-responsive nanosystems encompass pH-responsive NPs, for instance, by incorporating the surfactant 77 KL ( N α , N ε -dioctanoyl lysine with a lithium counterion) [ 47 ], redox-responsive NPs, like those based on L -cysteine covalently linked to CS [ 47 ], and light-responsive NPs, functionalized with photocatalytic titanium dioxide NPs [ 48 ]. These innovative formulations, often utilizing modified forms of CS, aim to enhance the targeting capability of MTX-loaded CS NPs and exert increased control over the release of the chemotherapeutic agent. While many studies showcase the improved efficiency of MTX-loaded CS-based NPs, a notable limitation is the lack of comprehensive preclinical data in several of these investigations. Future research should aim to bridge this gap, providing a more robust foundation for the translation of these findings into clinical applications. Furthermore, magnetically driven NPs can be advantageous in achieving targeted delivery to tumor cells and other disorders [ 50 – 52 ]. Magnetite (Fe 3 O 4 ) stands out as a distinctive category of magnetic nanoparticles that has garnered considerable attention the medical field. Due to their biocompatibility, extensive chemical affinity and distinctive magnetic properties, Fe 3 O 4 NPs have found widespread use in biomedical applications [ 53 ]. A primary application within the medical domain is evident in magnetic resonance imaging, where the superparamagnetic characteristics of Fe 3 O 4 contribute to enhanced imaging contrast, facilitating clearer and more detailed visualization of tissues and organs. In addition, Fe 3 O 4 particles play a crucial role in targeted drug delivery systems, enabling the precise attachment or encapsulation of drugs within these NPs for specific delivery to targeted cells or tissues [ 54 ]. This targeted strategy not only enhances the therapeutic effectiveness of drugs but also minimizes potential side effects. Despite their immense potential, the use of Fe 3 O 4 NPs in medical applications presents certain challenges. One notable hurdle involves concerns about potential toxicity, as the body’s response to iron-based NPs needs careful consideration. Another obstacle pertains to the susceptibility of Fe 3 O 4 to air oxidation, which can lead to the loss of magnetic properties. Maintaining the stability of Fe 3 O 4 particles in physiological environments and preventing aggregation are critical for successful medical applications. In addressing these challenges, researchers have directed their efforts toward surface modifications of Fe 3 O 4 NPs. Coating these particles with various materials, including natural substances such as CS or synthetic polymers like polyethylene glycol and polyvinylpyrrolidone, enhances their biocompatibility and stability. Furthermore, endeavors have been undertaken to optimize the size, shape, surface charge and functionalization of Fe 3 O 4 NPs, aiming to enhance their overall performance in Biomedicine [ 50 , 53 ]. When Fe 3 O 4 is incorporated within the CS matrix the resulting core/shell nanocomposite combines the magnetic responsiveness of Fe 3 O 4 NPs with the inherent benefits of CS [ 54 – 57 ]. These magnetically driven NPs have shown promise in targeted drug delivery, hyperthermia, and imaging applications. The magnetic responsiveness enables the NPs to be guided to specific sites within the body using external magnetic fields. This targeted approach enhances the precision of drug delivery, minimizing off-target effects and improving therapeutic outcomes. Furthermore, the combination of CS and Fe 3 O 4 in NPs offers opportunities for multifunctionality. Beyond drug delivery, these particles can be explored for applications such as magnetic hyperthermia, where the magnetic properties are harnessed to generate localized heat for therapeutic purposes [ 58 , 59 ]. In this study, we explore magnetically targeted and pH-responsive Fe 3 O 4 /CS (core/shell) nanocomposites as a drug delivery platform for MTX. The research delves into the formulation and characterization of these NPs, emphasizing their stability, MTX loading capacity and magnetic responsiveness. In addition, the potential pH-responsive MTX release behavior is investigated under the acidic microenvironment typical of tumors (pH ≈ 5.5). Furthermore, the hemocompatibility and cytotoxicity of these MTX-loaded magnetic nanocomposites against human breast cancer cells are thoroughly examined, providing crucial insights into their safety and efficacy profiles. To the best of our knowledge, this is the first time that these Fe 3 O 4 /CS (core/shell) NPs with dual targeted features, magnetic and pH responsiveness, have been developed as a platform for the delivery of MTX molecules to malignant cells. By combining the advantageous features of the developed NPs, this research aims to significantly contribute to the advancement of targeted MTX-based cancer therapy. 2. Materials and methods 2.1. Materials All chemicals were of analytical quality. Iron(III) chloride hexahydrate, iron(II) chloride tetrahydrate, hydrochloric acid, perchloric acid, and acetic acid were purchased from VWR International, LLC (Spain). Kolliphor® P-188 was purchased from BASF (Germany). CS (M W ≈ 50 to 190 kDa, 75–85% deacetylated), ammonia, citric acid (C 6 H 8 O 7 ), sodium sulfate, sodium citrate, sodium hydroxide (NaOH), sodium chloride (NaCl), disodium hydrogen phosphate (Na 2 HPO 4 ), ethylenediaminetetraacetic acid (EDTA), phosphate buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl tetrazolium bromide (MTT), MTX, dimethyl sulfoxide, Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), and Penicillin-Streptomycin solution (containing 10,000 U/mL of Penicillin and 10 mg/mL of Streptomycin) were purchased from Merck KGaA (Germany). Water was deionized and filtered with a Milli-Q Academic® system (Millipore, Spain). 2.2. Methods 2.2.1. Formulation of magnetite, chitosan, and magnetite/chitosan (core/shell) nanoparticles Fe 3 O 4 NPs were prepared using a controlled chemical co-precipitation process [52–55]. Initially, 40 mL of 2 M iron(III) chloride solution and 10 mL of 1 M iron(II) chloride solution (in 2 M hydrochloric acid) were gradually added to 500 mL of 1.5 M ammonia solution, under mechanical stirring (700 rpm; IKA® Eurostar 60 Digital Constant-Speed Mixer, Germany) at room temperature. After 30 min of continuous stirring, the resulting iron oxide particles were magnetically separated with a 0.4 T permanent magnet and re-dispersed in a 2 M nitric acid solution to achieve a stable aqueous dispersion. Following 1 h, the Fe 3 O 4 NPs were magnetically decanted again, followed by systematic cleaning through repeated magnetic separation and redispersion in water until the supernatant became transparent, and its conductivity indicated the absence of both unreacted chemicals and non-magnetic particles (achieved conductivity of the supernatant was ≤ 10 µS·cm − 1 ; Crison Microcm 2202 conductivity meter, Hach Lange Spain S.L.U., Spain). Subsequently, the NPs were dried at 60.0 ± 0.5 ºC in a convection oven (J.P. Selecta, S.A., Spain) and stored for later use. CS NPs were produced using a coacervation method, wherein the introduction of sodium sulfate into a solution of CS in acetic acid reduces the solubility of the polymer, leading to its rapid precipitation into NPs [38–40, 52, 54]. Specifically, a 1% (w/v) solution of CS was prepared in 50 mL of an aqueous solution of acetic acid (2%, v/v) containing 1% (w/v) Kolliphor® P-188, and the pH was adjusted to pH 4 with 1 M sodium hydroxide solution. Subsequently, 12.5 mL of a sodium sulfate solution (20%, w/v) was added drop-wise (at a rate of 2.5 mL/min) to the CS solution while mechanically stirring at 1,200 rpm. The stirring continued for 1 h to yield an aqueous dispersion of CS NPs. To purify the colloid, a cleaning process involving multiple cycles of centrifugation at 11,000 rpm for 60 min (Centrifuge 5804, Eppendorf Ibérica S.L.U., Spain) and redispersion in water was followed. This purification process was repeated until the conductivity of the supernatant reached ≤ 10 mS·cm − 1 . Magnetically responsive Fe 3 O 4 /CS (core/shell) NPs were produced using a coacervation method identical to the one employed for the CS particles described above. In the initial step of the formulation process, Fe 3 O 4 nuclei (0.75%, w/v) were introduced into the aqueous acetic acid solution used for CS dissolution. Subsequently, the NP dispersion was subjected to a magnetic purification procedure, where the resulting NPs were repetitively isolated from the liquid medium, using a 0.4 T permanent magnet, and redispersed in pure water until the supernatant’s conductivity reached ≤ 10 mS·cm − 1 . MTX was loaded to the magnetic core/shell NPs using an entrapment technique. The same procedure used for obtaining the CS-coated Fe 3 O 4 NPs was followed, where the anticancer agent was dissolved in the aqueous polymer solution of pH ≈ 4. After the preparation process, any excess of drug molecules was eliminated through magnetic cleaning, as described above. To study the effect of MTX concentration on its loading, drug concentrations ranging from 10 − 5 to 10 − 4 M were examined. 2.2.2. Characterization of size, surface electrical charge, and short-term stability The mean particle size and size distribution (polydispersity index, PdI) as well as the zeta potential ( ζ ) of the NPs were determined by photon correlation spectroscopy and electrokinetic determinations, respectively, after appropriate dilution of the aqueous colloids (≈ 0.1%, w/v). These measurements were performed using a Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK). The experiments were conducted at a constant cell temperature of 25.0 ± 0.5°C, and the detection angle employed was 60°. Furthermore, size and surface characteristics of the magnetic particles were examined using high resolution transmission electron microscopy (HRTEM), annular bright field scanning transmission electron microscopy (ABF-STEM) and high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) (Titan G2 60–300 FEI microscope, Thermofisher Scientific Inc., USA; operating at an accelerating voltage of 300 kV). Prior to observation, diluted aqueous NP dispersions (≈ 0.1%, w/v) were sonicated for 5 min, and droplets were placed on formvar/carbon-coated copper microgrids. Subsequently, the samples were dried in a convection oven (25.0 ± 0.5°C) (J. P. Selecta, S. A., Spain). Elemental analysis was conducted simultaneously with the TEM measurements using an energy dispersive X-ray (EDX) spectrometer (Bruker Nano GmbH, Germany). An assessment of the CS coating on Fe 3 O 4 NPs was conducted by analyzing the influence of pH and ionic strength on the ζ of the particles [39, 40, 58]. To assess the effect of pH, the study encompassed a range from pH 3 to 9, in the presence of 1 mM NaCl. Additionally, the impact of ionic strength was examined using various concentrations of NaCl while maintaining a constant pH ≈ 4. These determinations were carried out at room temperature ( n = 9) after 24 h of contact under stirring conditions (200 rpm, Boeco universal orbital shaker OS-10, Boeco, Germany). Moreover, the short-term stability of the Fe 3 O 4 /CS (core/shell) colloidal formulation (1 mg/mL, pH ≈ 6) was evaluated through incubation at 4.0 ± 0.5 ºC and 25.0 ± 0.5 ºC for a duration of 90 days. Throughout the experiment, the size, PdI and ζ values of the NPs were measured to monitor any changes in their properties. 2.2.3. Exploring magnetic properties The magnetic characteristics of the Fe 3 O 4 /CS (core/shell) particles were investigated using a Quantum Design MPMS XL (USA) SQUID magnetometer at room temperature. In addition, the magnetic responsiveness of the NPs was qualitatively assessed by observing a 0.1% (w/v) aqueous dispersion under a 0.4 T permanent magnet, using a Nikon SMZ800 stereoscopic zoom microscope (Nikon, Japan) [38, 59, 60]. 2.2.4. Quantification of Methotrexate loading and in vitro release The determination of the amount of drug loaded to the Fe 3 O 4 /chitosan NPs was carried out by UV spectrophotometric analyses of the MTX remaining in the supernatant after NP centrifugation (60 min at 11,000 rpm). The loaded drug quantity was calculated by subtracting the drug released into the medium from the total amount used during particle preparation. To account for possible absorbance contributions of other formulation components, e.g. the surfactant agent, the absorbance of the supernatant from blank NPs (containing no MTX) was subtracted. MTX concentration was measured at its maximum absorbance wavelength ( λ max = 304 nm) by Ultraviolet–Visible spectroscopy (UV–Vis Dinko spectrophotometer, Dinko, Spain). Finally, the drug content was expressed as entrapment efficiency (EE, %) and drug loading (DL, %) according to Equations 1 and 2. $$EE \left(\text{%}\right)=\frac{\text{t}\text{o}\text{t}\text{a}\text{l} \text{M}\text{T}\text{X} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \left(\text{m}\text{g}\right)-\text{u}\text{n}\text{e}\text{n}\text{c}\text{a}\text{p}\text{s}\text{u}\text{l}\text{a}\text{t}\text{e}\text{d} \text{M}\text{T}\text{X} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \left(\text{m}\text{g}\right)}{\text{t}\text{o}\text{t}\text{a}\text{l} \text{M}\text{T}\text{X} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \left(\text{m}\text{g}\right)} \times 100$$ 1 $$DL \left(\text{%}\right)= \frac{\text{t}\text{o}\text{t}\text{a}\text{l} \text{M}\text{T}\text{X} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \left(\text{m}\text{g}\right)-\text{u}\text{n}\text{e}\text{n}\text{c}\text{a}\text{p}\text{s}\text{u}\text{l}\text{a}\text{t}\text{e}\text{d} \text{M}\text{T}\text{X} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \left(\text{m}\text{g}\right)}{\text{t}\text{o}\text{t}\text{a}\text{l} \text{m}\text{a}\text{s}\text{s} \text{o}\text{f} \text{N}\text{P}\text{s} \left(\text{m}\text{g}\right)} \times 100$$ 2 Drug release from the CS-coated Fe 3 O 4 particles were studied in buffers at two distinct pH values: a C 6 H 8 O 7 -NaOH buffer at pH 5.5 ± 0.1 (pH of acidic microenvironment in tumors) and a C 6 H 8 O 7 -Na 2 HPO 4 buffer at pH 7.4 ± 0.1 (pH of bloodstream). The magnetic colloid, 2 mL of NPs containing 5 mg/mL of MTX, was dispersed in 200 mL of each buffer. Throughout the experiment, dispersions were maintained at 37.0 ± 0.5 ºC and under mechanical stirring (100 rpm). At predetermined time intervals, 1 mL of the release medium was withdrawn and the concentration of MTX was determined by UV–Vis spectroscopy at 304 nm. After each sampling, an equal volume of the release medium, maintained at 37.0 ± 0.5°C, was replenished to maintain the volume of the release medium and ensure sink conditions. The same analytical procedure used to determine the DL (%) was applied, and the in vitro MTX release was calculated using Eq. 3: $$\text{C}\text{u}\text{m}\text{u}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{M}\text{T}\text{X} \text{r}\text{e}\text{l}\text{e}\text{a}\text{s}\text{e} \left(\text{%}\right)= \frac{\text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} \text{M}\text{T}\text{X} \text{r}\text{e}\text{l}\text{e}\text{a}\text{s}\text{e}\text{d} \text{i}\text{n} \text{t}\text{h}\text{e} \text{m}\text{e}\text{d}\text{i}\text{u}\text{m} \left(\text{m}\text{g}\right) }{\text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} \text{M}\text{T}\text{X} \text{l}\text{o}\text{a}\text{d}\text{e}\text{d} \text{i}\text{n} \text{t}\text{h}\text{e} \text{N}\text{P}\text{s} \left(\text{m}\text{g}\right)} \times 100$$ 3 2.2.5. Ex vivo hemocompatibility The evaluation of the nanosystem’s hemocompatibility provides valuable insights into its potential clinical applications. Human blood, sourced from healthy donors, was collected into flasks containing EDTA (for hemolysis and platelet activation experiments) or sodium citrate (for complement system activation and plasma clotting time assays), and treated following established procedures [15, 61, 62]. PBS served as the negative control in these experiments. The developed particles were incubated during 24 h with blood aliquots to assess their impact on erythrocyte lysis (hemoglobin release), complement activation (C3a release), platelet activation (sP-selectin release), and plasma recalcification time (T 1/2 max), employing validated UV–Vis spectrophotometric methodologies. 2.2.6. In vitro cell culture experiments 2.2.6.1. Cell maintenance MCF-10A human breast epithelial cells and MCF-7 human breast cancer cells (American Type Culture Collection, USA) were cultured in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin solution, and were maintained in a humidified 5% CO 2 incubator at 37.0 ± 0.5 ºC until used in the experiments [MCO-19AIC(UV) CO 2 incubator, Sanyo, Japan]. 2.2.6.2. In vitro cytotoxicity assay The cytotoxicity of MTX-loaded NPs was assessed using the MTT proliferation assay, which determines mitochondrial dehydrogenase activity. Various concentrations of free MTX, blank NPs and MTX-loaded NPs, dissolved or dispersed in the culture medium, were added to the cells and incubated for 72 h at 37.0 ± 0.5 ºC in a humidified atmosphere with 5% CO 2 . Subsequently, MTT solution (20 µL/well, 5 mg/mL in cell culture medium) was added and the cells were further incubated for 3 h at 37.0 ± 0.5°C. After removal of the culture medium, formazan crystals were dissolved in 200 µL of dimethyl sulfoxide. The optical density (OD) of the resulting dye, proportional to the number of viable (metabolically active) cells, was measured at 570 nm (Dynatech MR7000 microplate reader, Dynatech Laboratories, Inc., USA). Cells treated with Triton ® X-100 (1%, v/v) or incubated with cell culture medium without treatment served as controls. The relative cell viability (RCV, %) was calculated using Eq. 4. $$\text{R}\text{C}\text{V} \left(\text{%}\right)= \frac{\text{O}\text{D} \text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d} \text{c}\text{e}\text{l}\text{l}\text{s} }{\text{O}\text{D} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \left(\text{u}\text{n}\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\right) \text{c}\text{e}\text{l}\text{l}\text{s}} \times 100$$ 4 The half maximal inhibitory concentration (IC 50 ) values were calculated through non-linear regression analysis using GraphPad Prism 9.1.0 software (GraphPad Software Inc., USA). 2.2.7. Statistical analysis The collected data were subjected to statistical analysis using the SPSS Statistics software package (version 26.0; IBM Corporation, USA). Student’s t -test was utilized for comparing results, ensuring a 95% confidence interval. Each experiment was conducted in three independent assays. The results were presented as mean values ± standard deviation (SD). Statistical significance was defined at p < 0.05. 3. Results 3.1. Size, surface electrical charge, and short-term stability The characteristics of Fe 3 O 4 /CS (core/shell) NPs are demonstrated in Table 1 (time 0 days). While the mean diameter of Fe 3 O 4 particles was ≈ 11 nm, it was remarkably increased when these iron oxide nuclei were surface coated with CS to ≈ 270 nm. In addition, the PdI also increased from ≈ 0.031 to ≈ 0.138 for unmodified and coated Fe 3 O 4 NPs, respectively. Table 1 Mean diameter (nm), PdI, and ζ data (mV ) of the Fe 3 O 4 /CS (core/shell) NPs as a function of time (days) at 4.0 ± 0.5 ºC or 25.0 ± 0.5 ºC. Experimental values are indicated as means ± SDs ( n = 3). Short-term stability assay at 25.0 ± 0.5 ºC Short-term stability assay at 4.0 ± 0.5 ºC Time (days) Size (nm) PdI ζ (mV) Time (days) Size (nm) PdI ζ (mV) 0 270.4 ± 9.1 0.138 ± 0.021 23.5 ± 2.1 0 270.4 ± 9.1 0.138 ± 0.021 23.5 ± 2.1 3 263.9 ± 7.1 0.142 ± 0.032 25.2 ± 3.2 3 279.6 ± 11.6 0.161 ± 0.023 24.1 ± 6.3 7 287.4 ± 4.7 0.126 ± 0.019 26.2 ± 6.4 7 260.3 ± 6.4 0.147 ± 0.018 28.5 ± 4.2 14 282.6 ± 5.3 0.131 ± 0.021 22.2 ± 4.9 14 289.2 ± 3.3 0.107 ± 0.016 24.4 ± 7.1 30 273.4 ± 7.3 0.141 ± 0.024 26.6 ± 5.1 30 294.6 ± 6.2 0.129 ± 0.022 23.4 ± 4.2 60 313.9 ± 11.4 0.163 ± 0.038 19.2 ± 3.7 60 339.1 ± 9.2 0.157 ± 0.026 19.6 ± 4.1 90 372.2 ± 23.8 0.191 ± 0.033 17.4 ± 4.9 90 391.3 ± 29.7 0.203 ± 0.043 17.3 ± 3.2 The core/shell structure and elemental composition of the CS-coated Fe 3 O 4 NPs were examined through electron microscopy and EDX mapping analysis (Fig. 1 ). The HRTEM image (Fig. 1 a), alongside HAADF-STEM (Fig. 1 b) and ABF-STEM images (Fig. 1 c) clearly illustrated the embedding of iron oxide nuclei within the polymeric matrices. The observed clustering of NPs was likely a consequence of the sample preparation method involving drying for microscopy observations, a phenomenon documented in existing literature [ 38 , 49 , 63 ]. EDX analyses further validated the NP composition, verifying the presence of Fe, C, N and O elements within the NPs (Fig. 1 h). Specifically, the Fe and O elements were associated with the Fe 3 O 4 cores, while the N and C elements originated from the CS shell. The detection of Cu and Si elements in the EDX analysis could be explained by the utilization of copper-based grids [ 60 ] and the secondary fluorescence generated and detected by the fluorescence detector [ 64 ]. Elemental mapping of Fe and O (Figs. 1 e and 1 g) highlighted the uniform distribution of Fe 3 O 4 nuclei within the NP matrix, while the mapping of C and N (Figs. 1 d and 1 f) demonstrated the effective coating of CS onto the Fe 3 O 4 nanostructures. To assess the coating efficiency of CS around the iron oxide particles, electrophoretic characteristics of the colloids were studied (Fig. 2 ). At pH 3, all NPs, including Fe 3 O 4 , CS and Fe 3 O 4 /CS NPs, displayed a positive ζ ranging from + 50 to + 60. With increasing pH, both CS and Fe 3 O 4 /CS NPs showed a gradual decrease in ζ , approaching electroneutrality at pH 9 (Fig. 2 a). Conversely, Fe 3 O 4 particles exhibited a steeper decline, reaching the isoelectric point, or pH of zero ζ , at pH ≈ 7, and dramatically dropping to ≈ -90 at pH 9. Furthermore, when the NaCl concentration (at pH ≈ 4) was raised from 10 − 5 M to 10 − 2 M, the ζ of Fe 3 O 4 /CS NPs increased from ≈ + 30 to ≈ + 40, similar to the trend observed with CS NPs (Fig. 2 b). In contrast, Fe 3 O 4 NPs showed minimal variation in the ζ across the tested range of ionic strengths. Finally, short-term stability of aqueous dispersion of the Fe 3 O 4 /CS (core/shell) NPs at both 4 ºC and 25 ºC demonstrated relatively stable particle size and surface electrical charge (Table 1 ). The NPs showed high stability under the used storage conditions, both 4 ºC and 25 ºC, over the first 30 days as can be observed by the minimal changes in their characteristics. However, when the particles were further stored up to 90-day, they were found to grow in size by ≈ 1.4-fold, under both storage conditions. A similar trend was also observed with the PdI values which increased from ≈ 0.14 to ≈ 0.19. 3.2. Magnetic responsiveness The magnetic responsiveness of the Fe 3 O 4 /CS NPs was investigated by analyzing the hysteresis cycle, as shown in Fig. 3 a. The Figure clearly illustrates the nanocomposite’s soft magnetic character, as the increasing and decreasing field branches of the hysteresis cycle are hardly distinguishable. By examining the linear portions (low field) of the curve, it was possible to estimate the initial susceptibility as (0.24 ± 0.03) × 10 − 3 m 3 /Kg, and the saturation magnetization as 19.13 ± 1.06 Am 2 /Kg for the NPs. The effective magnetic response of the core/shell particles was further qualitatively confirmed by observing the colloid’s behavior when exposed to a permanent magnet (Fig. 3 b). Notably, the NPs exhibited complete magnetic attraction towards a 0.4 T magnet within 45 s. Additionally, the assessment of the nanocomposite’s magnetic responsiveness was extended through optical microscope visualization of the colloid under the influence of the magnet (Fig. 3 c). Initially, the aqueous dispersion of particles was uniformly distributed. However, upon exposure to the magnetic field, significant changes occurred. Chainlike aggregates formed parallel to the field lines, providing visual evidence of the NPs’ magnetic responsiveness. 3.3. Methotrexate loading and release The EE (%) and DL (%) of MTX in the CS-coated Fe 3 O 4 particles are detailed in Table 2 . With an increase in drug concentration during NP preparation, ranging from 10 − 5 to 10 − 4 M, the drug loading capacity experienced significant increments. Specifically, EE (%) and DL (%) values increased substantially from ≈ 5% and ≈ 0.009% to ≈ 36% and ≈ 0.65%, respectively ( p < 0.05). Table 2 Loading of Methotrexate (EE and DL, %) to the Fe 3 O 4 /CS (core/shell) particles. Experimental values are indicated as means ± SDs ( n = 3). [Methotrexate] (M) EE (%) DL (%) 10 − 5 5.1 ± 0.9 0.009 ± 0.002 3 × 10 − 5 15.1 ± 0.9 0.082 ± 0.005 5 × 10 − 5 21.9 ± 1.8 0.199 ± 0.016 7 × 10 − 5 29.4 ± 2.1 0.374 ± 0.027 10 − 4 35.8 ± 2.2 0.651 ± 0.039 The pH-responsive MTX release from the Fe 3 O 4 /CS (core/shell) NPs was assessed at 37.0 ± 0.5 ºC by using release media stimulating the pH conditions of the bloodstream and the acidic environment in the endosomes and lysosomes of tumor cells (Fig. 4 ). At pH ≈ 7.4, a distinct biphasic drug release profile was observed, characterized by an initial rapid burst release of ≈ 60% of MTX within the first 6 h. Subsequently, the remaining chemotherapeutic was released at a slower rate over the next 90 h. In contrast, at pH ≈ 5.5, a significantly faster drug release rate (≈ 2.9-fold) was noted, leading to complete MTX release achieved within only 3 h ( p < 0.05). 3.4. Ex vivo hemocompatibility The blood compatibility tests conducted on blank core/shell particles are summarized in Table 3 . These magnetic nanocomposites exhibited minimal erythrolytic activity even after 24 h of incubation, with only ≈ 2% hemolysis observed. Furthermore, the analysis of complement system activation, platelet activation and plasma clotting times indicated that the Fe 3 O 4 /CS NPs had no significant impact on related parameters such as sP-selectin release, C3a release and T 1/2 max . Table 3 Effect of the Fe 3 O 4 /CS (core/shell) NPs on hemolysis (%), complement activation (C3a release: C3a desArg, ng/mL), platelet activation (sP-selectin release, ng/mL), and plasma recalcification time (T 1/2 max , min). Data is expressed as means ± SDs ( n = 3). Fe 3 O 4 /CS NPs Control (PBS solution) Hemolysis (%) Incubation time: 2 h 2.43 ± 0.63 0 Incubation time: 6 h 2.15 ± 0.75 0 Incubation time: 12 h 1.91 ± 0.44 0 Incubation time: 24 h 2.07 ± 0.62 0 C3a desArg (ng/mL) 299.7 ± 7.6 301.4 ± 4.2 sP-selectin release (ng/mL) 101.3 ± 3.5 102.2 ± 3.4 T 1/2 max (min) 11.4 ± 2.6 10.3 ± 3.1 3.5. In vitro cytotoxicity assay The results of the cytotoxic evaluation of Fe 3 O 4 /CS particles in MCF-10A breast epithelial cells and MCF-7 human breast cancer cells after 72 h of incubation are presented in Fig. 5 . It is evident that increasing concentrations of the blank core/shell NPs (from 5 to 200 µM) induced minimal cytotoxicity in both cell lines (Fig. 5 a). Figure 5 b demonstrates a dose-dependent inhibition of cancer cell growth observed with MTX treatment, both free and loaded to the NPs. In comparison with free drug, MTX-loaded magnetic particles significantly enhanced the MTX antitumor activity at concentrations ranging from 10 to 100 µg/mL ( p < 0.05). Furthermore, the IC 50 of the MTX-loaded core/shell NPs (26.4 ± 2.1 µg/mL) was ≈ 2.7-fold less than that of the free anticancer drug (72.3 ± 1.6 µg/mL) ( p < 0.05). 4. Discussion In this study, magnetically responsive Fe 3 O 4 /CS particles loaded with the chemotherapeutic MTX were developed and extensively characterized. The formulated NPs demonstrated good loading capacity for the anticancer therapeutic agent and promising characteristics for possible biomedical applications. Thus, their potential as a targeted drug delivery system for cancer treatment was explored using various in vitro experiments. Building on previous research, the preparation of the core/shell NPs was convincingly demonstrated through the effective and uniform embedding of iron oxide nuclei within the CS polymeric coating. This was confirmed through the morphological analysis under the electron microscope and elemental mappings via EDX. The formulation method involved initially obtaining Fe 3 O 4 particles, followed by chitosan coacervation around the Fe 3 O 4 nuclei, a widely employed procedure for creating CS-coated magnetic NPs [ 38 , 39 , 52 , 54 , 56 , 63 ]. Although CS surface functionalization of the Fe 3 O 4 NPs is associated with an increase in size (from ≈ 11 nm to ≈ 270 nm), it imparts a hydrophilic positive surface charge that may protect iron oxide particles from capture by the mononuclear phagocytic system, thus probably preventing rapid clearance from the systemic circulation and making the nanocomposites suitable for parenteral administration. Consequently, these stealth properties, combined with the appropriate size range, may enhance the NPs’ plasma half-lives, facilitating their delivery to the tumor site and extravasation into tissues through the gaps between endothelial cells of the tumor vasculature (up to ≈ 600 nm), capitalizing on the enhanced permeability and retention effect [ 38 , 49 , 65 ]. Moreover, the positive surface charge conferred by CS prevents particle aggregation, thanks to the electrostatic repulsion generated. This, in turn, ensures the physical stability of the NPs when in aqueous dispersion. Notably, short-term stability analysis conducted in this study confirmed the efficacy of the preparation method in formulating well-stabilized core/shell NPs. In fact, the nanocomposites maintained a stable size and surface electrical charge, with no observable aggregation for at least 30 days when stored at both 4 ºC and 25 ºC (Table 1 ). These findings were also confirmed when the electrophoretic properties of the NPs were examined as a function of pH and ionic strength. CS-coated NPs exhibited a positive ζ between + 40 and + 50 at pH ≈ 5, indicating their stability and potential for controlled release under acidic conditions, such as in the tumor microenvironment. The behavior of Fe 3 O 4 /CS NPs in the presence of varying ionic strengths further highlighted their stability and suitability for biological applications. In biological settings, positively charged NPs exhibit an ability to interact with negatively charged mucus and cells, leading to improved permeation, absorption and bioavailability of the particles and their cargo. In addition, the mucoadhesive characteristics exhibited by CS-coated NPs may play a role in prolonging the drug’s action by extending their residence time at mucous-rich sites. Moreover, these positive surface electrical charges could potentially enhance the internalization of NPs by negatively charged cancer cells, ensuring the delivery of the therapeutic molecule to the intracellular targets [ 35 , 37 , 66 – 68 ]. Furthermore, the incorporation of MTX, which is a poly-functional weak dicarboxylic acid (pKa values of 3.8, 4.8 and 5.6) [ 69 ], into the CS matrix could be the consequence of electrostatic attractions between drug molecules, negatively charged when the –COOH groups are protonated, and the positively charged polymer ( ≈ + 40 mV at pH 4). The electrostatic interaction between MTX and CS also influences the drug release profile resulting in a pH-responsive MTX release behavior. While under pH ≈ 7.4, MTX shows a sustained release over the next 90 h, the chemotherapeutic is completely released within 3 h under acidic conditions (pH 5.5). The observed pH-responsiveness, as previously documented in studies involving CS-coated NPs [ 38 , 40 ], may be attributed to the presence of the hydrophilic CS coating. With a pKa of 6.5, the amine groups of CS undergo ionization in acidic solutions, making it more soluble at lower pH levels. This, in turn, accelerates drug diffusion and release [ 36 , 70 ]. This behavior aligns with the targeted drug delivery requirements, ensuring preferential drug release in the acidic tumor microenvironment, thereby enhancing therapeutic efficacy while minimizing systemic side effects. The magnetic responsiveness of the Fe 3 O 4 /CS NPs was demonstrated through hysteresis cycle analysis and visual observations under the influence of a magnetic field. The soft magnetic nature of the nanocomposites was evident, and their rapid response to a magnetic field, forming chainlike aggregates parallel to the field lines, indicated their potential for targeted drug delivery [ 38 , 39 , 54 ]. This could result from the notable contribution of the magnetic interaction over the Derjaguin-Landau-Verwey-Overbeek (DLVO) colloidal interactions between the NPs ( e.g. electrostatic van der Waals and hydration or acid–base) [ 49 ]. However, in vivo experiments should be done to clarify if this magnetic responsiveness could determine the accumulation of the CS-coated Fe 3 O 4 NPs at a targeted site. In the ex vivo and in vitro biocompatibility assessments, the core/shell particles exhibited excellent hemocompatibility, and no significant impact on blood components. In addition, these NPs showed no in vitro cytotoxic effects on both normal and cancer cells. These results underscore the biocompatibility and safety of the nanocomposites, suggesting their potential for intravenous administration without inducing significant adverse effects. Lastly, MTX-loaded NPs exhibited a dose-dependent inhibition of cancer cell growth, significantly enhancing the antitumor activity compared to free MTX. The substantially lower IC 50 of the MTX-loaded NPs compared to the free drug, down by ≈ 2.7-fold, indicated a significant enhancement in the therapeutic efficacy of the formulated particles. These findings support the potential of Fe 3 O 4 /CS NPs as a promising platform for targeted delivery of MTX in cancer therapy, warranting further investigations into their in vivo safety and efficacy. 5. Conclusions In this research, magnetically targeted MTX-loaded Fe 3 O 4 /CS (core/shell) NPs were developed and thoroughly characterized. These particles exhibited favorable characteristics, including effective CS coating, stability, magnetic responsiveness, good drug loading capacity and a pH-responsive MTX release behavior, indicating their potential for targeted cancer therapy. These NPs displayed stable properties over at least 30 days, making them suitable for storage and transportation. Importantly, they showed excellent compatibility with blood components, suggesting their safe intravenous administration. In cytotoxicity tests, these nanocomposites demonstrated enhanced efficacy against breast cancer cells compared to free MTX. These findings collectively highlight their potential for clinical translation. Further in vivo studies are warranted to validate their efficacy, biodistribution and safety profile, paving the way for their application in personalized and targeted cancer treatment strategies. Declarations Ethical statement Ethics approval and consent to participate Not applicable. Consent for publication All the authors read and approved the final version of the manuscript. Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests. Funding This work was supported by FEDER, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i), Instituto de Salud Carlos III (FIS, Spain) (grant number PI19/01478), and FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Spain (Grant No. P20_00346). Authors' contributions Ana Medina-Moreno: data curation, formal analysis, investigation, methodology. Mazen M. El-Hammadi: formal analysis, validation, writing - original draft, writing - review & editing. Gema I. Martínez-Soler: data curation, formal analysis, investigation, methodology. Javier G. Ramos: data curation, formal analysis. Gracia García-García: data curation, formal analysis. José L. Arias: conceptualization, data curation, methodology, supervision, validation, writing - review & editing. Acknowledgements Not applicable. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References Kozminski P, Halik PK, Chesori R, Gniazdowska E. Overview of dual-acting drug methotrexate in different neurological diseases, autoimmune pathologies and cancers. Int J Mol Sci. 2020;21:3483. doi: 10.3390/ijms21103483. Cronstein BN, Aune TM. Methotrexate and its mechanisms of action in inflammatory arthritis. Nat Rev Rheumatol. 2020;16:145–54. doi: 10.1038/s41584-020-0373-9. Abolmaali SS, Tamaddon AM, Dinarvand R. A review of therapeutic challenges and achievements of methotrexate delivery systems for treatment of cancer and rheumatoid arthritis. Cancer Chemother Pharmacol. 2013;71:1115–30. doi: 10.1007/s00280-012-2062-0. Kumar S, Garg NK, Jain A, Khopade A, Pandey P, Sawant KK. Nanocarriers mediated delivery of methotrexate is instrumental in treating auto-immune diseases and cancer. J Drug Deliv Sci Technol. 2023;88:104969. doi: 10.1016/j.jddst.2023.104969. Solomon DH, Glynn RJ, Karlson EW, Lu F, Corrigan C, Colls J, Xu C, MacFadyen J, Barbhaiya M, Berliner N, Dellaripa PF, Everett BM, Pradhan AD, Hammond SP, Murray M, Rao DA, Ritter SY, Rutherford A, Sparks JA, Stratton J, Suh DH, Tedeschi SK, Vanni KMM, Paynter NP, Ridker PM. Adverse effects of low-dose methotrexate: a randomized trial. Ann Intern Med. 2020;172:369–80. doi: 10.7326/M19-3369. Mukhtar M, Ezra Manicum AL, Shojaei Barjouei M, Eshaghi Malekshah R, Behzadmehr R, Rahdar A, Ghotekar S, Baino F. Nanocarriers for methotrexate delivery/codelivery in the frame of cancer diagnostics and treatment: a review. Front Biomater Sci. 2023;2:1200670. doi: 10.3389/fbiom.2023.1200670. Giri BR, Yang HS, Song IS, Choi HG, Cho JH, Kim DW. Alternative methotrexate oral formulation: enhanced aqueous solubility, bioavailability, photostability, and permeability. Pharmaceutics. 2022;14:2073. doi: 10.3390/pharmaceutics14102073. Ray S, Joy M, Sa B, Ghosh S, Chakraborty J. pH dependent chemical stability and release of methotrexate from a novel nanoceramic carrier. RSC Adv. 2015;5:39482–94. doi: 10.1039/C5RA03546E. Shariatifar H, Ranjbarian F, Hajiahmadi F, Farasat A. A comprehensive review on methotrexate containing nanoparticles; an appropriate tool for cancer treatment. Mol Biol Rep. 2022;49:11049–60. doi: 10.1007/s11033-022-07782-7. Gavas S, Quazi S, Karpinski TM. Nanoparticles for cancer therapy: current progress and challenges. Nanoscale Res Lett. 2021;16:173. doi: 10.1186/s11671-021-03628-6. Nirmala MJ, Kizhuveetil U, Johnson A, Balaji G, Nagarajan R, Muthuvijayan V. Cancer nanomedicine: a review of nano-therapeutics and challenges ahead. RSC Adv. 2023;13:8606–29. doi: 10.1039/d2ra07863e. Sun L, Liu H, Ye Y, Lei Y, Islam R, Tan S, Tong R, Miao YB, Cai L. Smart nanoparticles for cancer therapy. Signal Transduct Target Ther. 2023;8:418. doi: 10.1038/s41392-023-01642-x. El-Hammadi MM, Arias JL. Advanced engineering approaches in the development of PLGA-based nanomedicines. In: Aliofkhazraei M, editor. Handbook of Nanoparticles. Cham: Springer International Publishing AG; 2015. pp. 1009–39. Rebollo R, Oyoun F, Corvis Y, El-Hammadi MM, Saubamea B, Andrieux K, Mignet N, Alhareth K. Microfluidic manufacturing of liposomes: development and optimization by design of experiment and machine learning. ACS Appl Mater Interfaces. 2022;14:39736–45. doi: 10.1021/acsami.2c06627. Jiménez-López J, El-Hammadi MM, Ortiz R, Cayero-Otero MD, Cabeza L, Perazzoli G, Martin-Banderas L, Baeyens JM, Prados J, Melguizo C. A novel nanoformulation of PLGA with high non-ionic surfactant content improves in vitro and in vivo PTX activity against lung cancer. Pharmacol Res. 2019;141:451–65. doi: 10.1016/j.phrs.2019.01.013. El-Hammadi MM, Delgado ÁV, Melguizo C, Prados JC, Arias JL. Folic acid-decorated and PEGylated PLGA nanoparticles for improving the antitumour activity of 5-fluorouracil. Int J Pharm. 2017;516:61–70. doi: 10.1016/j.ijpharm.2016.11.012. Cabeza L, El-Hammadi MM, Ortiz R, Cayero-Otero MD, Jiménez-López J, Perazzoli G, Martin-Banderas L, Baeyens JM, Melguizo C, Prados J. Evaluation of poly (lactic-co-glycolic acid) nanoparticles to improve the therapeutic efficacy of paclitaxel in breast cancer. BioImpacts. 2022;12:515–31. doi: 10.34172/bi.2022.23433. El-Hammadi MM, Small-Howard AL, Fernández-Arévalo M, Martín-Banderas L. Development of enhanced drug delivery vehicles for three cannabis-based terpenes using poly(lactic-co-glycolic acid) based nanoparticles. Ind Crop Prod. 2021;164:113345. doi: 10.1016/j.indcrop.2021.113345. El-Hammadi MM, Small-Howard AL, Jansen C, Fernandez-Arevalo M, Turner H, Martin-Banderas L. Potential use for chronic pain: poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles enhance the effects of cannabis-based terpenes on calcium influx in TRPV1-expressing cells. Int J Pharm. 2022;616:121524. doi: 10.1016/j.ijpharm.2022.121524. El-Hammadi MM, Arias JL. Nanotechnology for vaginal drug delivery and targeting. In: Mozafari M, editor. Nanoengineered Biomaterials for Advanced Drug Delivery. Amsterdam: Elsevier Ltd.; 2020. pp. 647–82. Bhatia SN, Chen X, Dobrovolskaia MA, Lammers T. Cancer nanomedicine. Nat Rev Cancer. 2022;22:550–6. doi: 10.1038/s41568-022-00496-9. El-Hammadi MM, Arias JL. Nanomedicine for vaginal drug delivery. In: Kesharwani P, Taurin S, Greish K, editors. Theory and Applications of Nonparenteral Nanomedicines. London: Academic Press: 2021. pp. 235–57. El-Hammadi MM, Arias JL. Recent advances in the surface functionalization of PLGA-based nanomedicines. Nanomaterials (Basel). 2022;12:354. doi: 10.3390/nano12030354. Madani F, Esnaashari SS, Bergonzi MC, Webster TJ, Younes HM, Khosravani M, Adabi M. Paclitaxel/methotrexate co-loaded PLGA nanoparticles in glioblastoma treatment: formulation development and in vitro antitumor activity evaluation. Life Sci. 2020;256:117943. doi: 10.1016/j.lfs.2020.117943. Jang JH, Jeong SH, Lee YB. Preparation and in vitro/in vivo characterization of polymeric nanoparticles containing methotrexate to improve lymphatic delivery. Int J Mol Sci. 2019;20:3312. doi: 10.3390/ijms20133312. Vakilinezhad MA, Amini A, Dara T, Alipour S. Methotrexate and curcumin co-encapsulated PLGA nanoparticles as a potential breast cancer therapeutic system: in vitro and in vivo evaluation. Colloids Surf B Biointerfaces. 2019;184:110515. doi: 10.1016/j.colsurfb.2019.110515. Noreen S, Hasan S, Ghumman SA, Bukhari SNA, Ijaz B, Hameed H, Iqbal H, Aslam A, Elsherif MAM, Noureen S, Ejaz H. pH responsive Abelmoschus esculentus mucilage and administration of methotrexate: in-vitro antitumor and in-vivo toxicity evaluation. Int J Mol Sci. 2022;23:2725. doi: 10.3390/ijms23052725. Barbosa AI, Costa Lima SA, Reis S. Development of methotrexate loaded fucoidan/chitosan nanoparticles with anti-inflammatory potential and enhanced skin permeation. Int J Biol Macromol. 2019;124:1115–22. doi: 10.1016/j.ijbiomac.2018.12.014. Saraei M, Sarvari R, Massoumi B, Agbolaghi S. Co-delivery of methotrexate and doxorubicin via nanocarriers of star-like poly(DMAEMA-block-HEMA-block-AAc) terpolymers. Polym Int. 2019;68:1795–803. doi: 10.1002/pi.5890. Bashir SM, Ahmed Rather G, Patrício A, Haq Z, Sheikh AA, Shah MZUH, Singh H, Khan AA, Imtiyaz S, Ahmad SB, Nabi S, Rakhshan R, Hassan S, Fonte P. Chitosan nanoparticles: a versatile platform for biomedical applications. Materials (Basel). 2022;15:6521. doi: 10.3390/ma15196521. Desai N, Rana D, Salave S, Gupta R, Patel P, Karunakaran B, Sharma A, Giri J, Benival D, Kommineni N. Chitosan: a potential biopolymer in drug delivery and biomedical applications. Pharmaceutics. 2023;15:1313. doi: 10.3390/pharmaceutics15041313. Harugade A, Sherje AP, Pethe A. Chitosan: a review on properties, biological activities and recent progress in biomedical applications. React Funct Polym. 2023;191:105634. doi: 10.1016/j.reactfunctpolym.2023.105634. Puluhulawa LE, Joni IM, Elamin KM, Mohammed AFA, Muchtaridi M, Wathoni N. Chitosan-hyaluronic acid nanoparticles for active targeting in cancer therapy. Polymers (Basel). 2022;14:3410. doi: 10.3390/polym14163410. Geethakumari D, Bhaskaran Sathyabhama A, Raji Sathyan K, Mohandas D, Somasekharan JV, Thavarool Puthiyedathu S. Folate functionalized chitosan nanoparticles as targeted delivery systems for improved anticancer efficiency of cytarabine in MCF-7 human breast cancer cell lines. Int J Biol Macromol. 2022;199:150–61. doi: 10.1016/j.ijbiomac.2021.12.070. Sharifi-Rad J, Quispe C, Butnariu M, Rotariu LS, Sytar O, Sestito S, Rapposelli S, Akram M, Iqbal M, Krishna A, Kumar NVA, Braga SS, Cardoso SM, Jafernik K, Ekiert H, Cruz-Martins N, Szopa A, Villagran M, Mardones L, Martorell M, Docea AO, Calina D. Chitosan nanoparticles as a promising tool in nanomedicine with particular emphasis on oncological treatment. Cancer Cell Int. 2021;21:318. doi: 10.1186/s12935-021-02025-4. Fernández-Álvarez F, García-García G, Arias JL. A tri-stimuli responsive (maghemite/PLGA)/chitosan nanostructure with promising applications in lung cancer. Pharmaceutics. 2021;13:1232. doi: 10.3390/pharmaceutics13081232. Mohammed MA, Syeda JTM, Wasan KM, Wasan EK. An overview of chitosan nanoparticles and its application in non-parenteral drug delivery. Pharmaceutics. 2017;9:53. doi: 10.3390/pharmaceutics9040053. Aibani N, Rai R, Patel P, Cuddihy G, Wasan EK. Chitosan nanoparticles at the biological interface: implications for drug delivery. Pharmaceutics. 2021;13:1686. doi: 10.3390/pharmaceutics13101686. Fernández-Álvarez F, García-García G, Arias JL. A tri-stimuli responsive (maghemite/PLGA)/chitosan nanostructure with promising applications in lung cancer. Pharmaceutics. 2021;13:1232. doi: 10.3390/pharmaceutics13081232. Fernández-Álvarez F, Caro C, García-García G, García-Martín ML, Arias JL. Engineering of stealth (maghemite/PLGA)/chitosan (core/shell)/shell nanocomposites with potential applications for combined MRI and hyperthermia against cancer. J Mater Chem B. 2021;9:4963–80. doi: 10.1039/D1TB00354B. Medina-Moreno A, El-Hammadi MM, Arias JL. pH-dependent, extended release and enhanced in vitro efficiency against colon cancer of Tegafur formulated using chitosan-coated poly(ε-caprolactone) nanoparticles. J Drug Deliv Sci Technol. 2023;86:104594. doi: 10.1016/j.jddst.2023.104594. Amoozgar Z, Park J, Lin Q, Yeo Y. Low molecular-weight chitosan as a pH-sensitive stealth coating for tumor-specific drug delivery. Mol Pharm. 2012;9:1262. doi: 10.1021/mp2005615. Ishak RA, Awad GA, Zaki NM, El-Shamy Ael H, Mortada ND. A comparative study of chitosan shielding effect on nano-carriers hydrophilicity and biodistribution. Carbohydr Polym. 2013;94:669–76. doi: 10.1016/j.carbpol.2013.01.072. Baati T, Chaabani I, Salek A, Njim L, Selmi M, Al-Kattan A, Hosni K. Chitosan-coated ultrapure silicon nanoparticles produced by laser ablation: biomedical potential in nano-oncology as a tumor-targeting nanosystem. Nanoscale Adv. 2023;5:3044–52. doi: 10.1039/d3na00253e. Elkomy MH, Ali AA, Eid HM. Chitosan on the surface of nanoparticles for enhanced drug delivery: a comprehensive review. J Control Release. 2022;351:923–40. doi: 10.1016/j.jconrel.2022.10.005. Nur SG, Buket O, Sezgi K, Cagdas A, Cansu A, Guliz A, Habibe Y, Cigir BA, Yavuz D, Senay HS. Synthesis of methotrexate loaded chitosan nanoparticles and in vitro evaluation of the potential in treatment of prostate cancer. Anticancer Agents Med Chem. 2016;16:1038–42. doi: 10.2174/1871520616666160101120040. Nogueira DR, Tavano L, Mitjans M, Perez L, Infante MR, Vinardell MP. In vitro antitumor activity of methotrexate via pH-sensitive chitosan nanoparticles. Biomaterials. 2013;34:2758–72. doi: 10.1016/j.biomaterials.2013.01.005. Mazzotta E, De Benedittis S, Qualtieri A, Muzzalupo R. Actively targeted and redox responsive delivery of anticancer drug by chitosan nanoparticles. Pharmaceutics. 2019;12:26. doi: 10.3390/pharmaceutics12010026. Al-Nemrawi N, Hameedat F, Al-Husein B, Nimrawi S. Photolytic controlled release formulation of methotrexate loaded in chitosan/TiO(2) nanoparticles for breast cancer. Pharmaceuticals (Basel). 2022;15:149. doi: 10.3390/ph15020149. Reddy LH, Arias JL, Nicolas J, Couvreur P. Magnetic nanoparticles: design and characterization, toxicity and biocompatibility, pharmaceutical and biomedical applications. Chem Rev. 2012;112:5818–78. doi: 10.1021/cr300068p. Materón Em, Miyazaki CM, Carr O, Joshi N, Picciani PHS, Dalmaschio CJ, Davis F, Shimizu FM. Magnetic nanoparticles in biomedical applications: a review. Appl Surf Sci Adv. 2021;6:100163. doi: 10.1016/j.apsadv.2021.100163. Fernández-Álvarez F, García-García G, Iglesias GR, Arias JL. A maghemite/PLGA (core/shell) nanostructure that may facilitate chemotherapy and antitumor hyperthermia. J Magn Magn Mater. 2024;589:171574. doi: 10.1016/j.jmmm.2023.171574. El-Hammadi MM, Arias JL. Iron oxide-based multifunctional nanoparticulate systems for biomedical applications: a patent review (2008 - present). Expert Opin Ther Pat. 2015;25:691–709. doi: 10.1517/13543776.2015.1028358. Arias JL, Reddy LH, Couvreur P. Fe3O4/chitosan nanocomposite for magnetic drug targeting to cancer. J Mater Chem. 2012;22:7622–32. doi: 10.1039/C2JM15339D. García-García G, Caro C, Fernández-Álvarez F, García-Martín ML, Arias JL. Multi-stimuli-responsive chitosan-functionalized magnetite/poly(epsilon-caprolactone) nanoparticles as theranostic platforms for combined tumor magnetic resonance imaging and chemotherapy. Nanomedicine. 2023;52:102695. doi: 10.1016/j.nano.2023.102695. Kazemi S, Pourmadadi M, Yazdian F, Ghadami A. The synthesis and characterization of targeted delivery curcumin using chitosan-magnetite-reduced graphene oxide as nano-carrier. Int J Biol Macromol. 2021;186:554–62. doi: 10.1016/j.ijbiomac.2021.06.184. Al-Hussainawy MK, Sahb Mehdi Z, Jasim KK, Alshamsi HA, Saud HR, Kyhoiesh HAK. A single rapid route synthesis of magnetite/chitosan nanocomposite: competitive study. Results Chem. 2022;4:100567. doi: 10.1016/j.rechem.2022.100567. Jamir M, Borgohain C, Borah JP. Chitosan modified Fe3O4 nanoparticles for hyperthermia application. Mater Today Proc. 2022;65:2484–9. doi: 10.1016/j.matpr.2022.04.176. García-García G, Lázaro-Callejón M, Fernández-Álvarez F, Iglesias GR, Arias JL. (Magnetite/poly(ε-caprolactone))/chitosan (core/shell)/shell nanocomposites with potential applications in hyperthermia cancer therapy. J Magn Magn Mater. 2023;588:171500. doi: 10.1016/j.jmmm.2023.171500. Moskvin M, Huntošová V, Herynek V, Matouš P, Michalcová A, Lobaz V, Zasońska B, Šlouf M, Seliga R, Horák D. In vitro cellular activity of maghemite/cerium oxide magnetic nanoparticles with antioxidant properties. Colloids Surf B Biointerfaces. 2021;204:111824. doi: 10.1016/j.colsurfb.2021.111824. Fang K, Song L, Gu Z, Yang F, Zhang Y, Gu N. Magnetic field activated drug release system based on magnetic PLGA microspheres for chemo-thermal therapy. Colloids Surf B Biointerfaces. 2015;136:712–20. doi: 10.1016/j.colsurfb.2015.10.014. García-García G, Fernández-Álvarez F, Cabeza L, Delgado ÁV, Melguizo C, Prados JC, Arias JL. Gemcitabine-loaded magnetically responsive poly(epsilon-caprolactone) nanoparticles against breast cancer. Polymers (Basel). 2020;12:2790. doi: 10.3390/polym12122790. Muñoz de Escalona M, Sáez-Fernández E, Prados JC, Melguizo C, Arias JL. Magnetic solid lipid nanoparticles in hyperthermia against colon cancer. Int J Pharm. 2016;504:11–9. doi: 10.1016/j.ijpharm.2016.03.005. Arias JL, López-Viota M, Sáez-Fernández E, Ruiz MA, Delgado ÁV. Engineering of an antitumor (core/shell) magnetic nanoformulation based on the chemotherapy agent ftorafur. Colloids Surf A Physicochem Eng Aspects. 2011;384:157–63. doi: 10.1016/j.colsurfa.2011.03.051. Santos DP, Ruiz MA, Gallardo V, Zanoni MVB, Arias JL. Multifunctional antitumor magnetite/chitosan-l-glutamic acid (core/shell) nanocomposites. J Nanopart Res. 2011;13:4311–23. doi: 10.1007/s11051-011-0378-z. Nasrazadani S, Hassani S. Modern analytical techniques in failure analysis of aerospace, chemical, and oil and gas industries. In: Makhlouf ASH, Aliofkhazraei M, editors. Handbook of Materials Failure Analysis with Case Studies from the Oil and Gas Industry. Amsterdam: Elsevier B.V. 2016. pp. 39–54. Wang J, Sui M, Fan W. Nanoparticles for tumor targeted therapies and their pharmacokinetics. Curr Drug Metab. 2010;11:129–41. doi: 10.2174/138920010791110827. He C, Hu Y, Yin L, Tang C, Yin C. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials. 2010;31:3657–66. doi: 10.1016/j.biomaterials.2010.01.065. Voon SH, Tiew SX, Kue CS, Lee HB, Kiew LV, Misran M, Kamkaew A, Burgess K, Chung LY. Chitosan-coated poly(lactic-co-glycolic acid)-diiodinated boron-dipyrromethene nanoparticles improve tumor selectivity and stealth properties in photodynamic cancer therapy. J Biomed Nanotechnol. 2016;12:1431–52. doi: 10.1166/jbn.2016.2263. Moraru C, Mincea M, Menghiu G, Ostafe V. Understanding the factors influencing chitosan-based nanoparticles-protein corona interaction and drug delivery applications. Molecules. 2020;25:4758. doi: 10.3390/molecules25204758. Wu P, He X, Wang K, Tan W, He C, Zheng M. A novel methotrexate delivery system based on chitosan-methotrexate covalently conjugated nanoparticles. J Biomed Nanotechnol. 2009;5:557–64. doi: 10.1166/jbn.2009.1073. Badran MM, Mady MM, Ghannam MM, Shakeel F. Preparation and characterization of polymeric nanoparticles surface modified with chitosan for target treatment of colorectal cancer. Int J Biol Macromol. 2017;95:643–49. doi: 10.1016/j.ijbiomac.2016.11.098. Supplementary Files HighlightsMtezSoler.docx GraphicalAbstractMtezSoler.tif Cite Share Download PDF Status: Published Journal Publication published 05 Sep, 2024 Read the published version in Drug Delivery and Translational Research → Version 1 posted Editorial decision: Major Revisions Needed 01 Jun, 2024 Reviewers agreed at journal 01 May, 2024 Reviewers invited by journal 01 May, 2024 Editor assigned by journal 30 Apr, 2024 First submitted to journal 29 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4328624","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":297690590,"identity":"99d1fb8a-3d64-4564-bdc7-2bb094397484","order_by":0,"name":"Ana Medina-Moreno","email":"","orcid":"","institution":"University of Granada: Universidad de Granada","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"","lastName":"Medina-Moreno","suffix":""},{"id":297690591,"identity":"b595b246-a5fe-424f-ba26-b726b84faf39","order_by":1,"name":"Mazen M. El-Hammadi","email":"","orcid":"","institution":"University of Seville: Universidad de Sevilla","correspondingAuthor":false,"prefix":"","firstName":"Mazen","middleName":"M.","lastName":"El-Hammadi","suffix":""},{"id":297690592,"identity":"39cea96e-59fb-4438-b0c2-e4e18f5a91df","order_by":2,"name":"Gema I. Martínez-Soler","email":"","orcid":"","institution":"University of Granada: Universidad de Granada","correspondingAuthor":false,"prefix":"","firstName":"Gema","middleName":"I.","lastName":"Martínez-Soler","suffix":""},{"id":297690593,"identity":"9d257d37-66a1-4033-94a5-d59915a36f00","order_by":3,"name":"Javier G. Ramos","email":"","orcid":"","institution":"University of Granada: Universidad de Granada","correspondingAuthor":false,"prefix":"","firstName":"Javier","middleName":"G.","lastName":"Ramos","suffix":""},{"id":297690594,"identity":"5a0e2b06-8913-41c3-9c36-a8df1fb4b673","order_by":4,"name":"Gracia García-García","email":"","orcid":"","institution":"University of Almeria: Universidad de Almeria","correspondingAuthor":false,"prefix":"","firstName":"Gracia","middleName":"","lastName":"García-García","suffix":""},{"id":297690595,"identity":"efebc5c1-22ef-4f90-a9e6-e8ddde4539be","order_by":5,"name":"Jose L. Arias","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYBACxnYgkcDAIEeClmaIFmMSrGGGUIkNxOtoZn724UFNXfqG82sfMHz4Q5TD2IxnJBw7nLvhxnMDxpltRGkBeiOB7QBQyzEGZl5inMfYzP6ZIeFfXboBSMsf4hzGY8yQ2MacYHC+DRgUbMRpKWZI7DtsOPMGG8PBXmL8Ytjevpnxx7c6eb7zxxgf/CDGYYYNMJZEAsMBIjQwMMjDWfzEaRgFo2AUjIIRCAASjDZPOEHMPQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3437-3791","institution":"University of Granada: Universidad de Granada","correspondingAuthor":true,"prefix":"","firstName":"Jose","middleName":"L.","lastName":"Arias","suffix":""}],"badges":[],"createdAt":"2024-04-26 09:31:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4328624/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4328624/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13346-024-01701-y","type":"published","date":"2024-09-05T16:04:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56275932,"identity":"088675cd-f250-481a-9148-66e98efc6ee1","added_by":"auto","created_at":"2024-05-10 20:07:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":537189,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cem\u003ea\u003c/em\u003e) HRTEM, (\u003cem\u003eb\u003c/em\u003e) ABF-STEM, and (\u003cem\u003ec\u003c/em\u003e) HAADF-STEM images of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) particles; EDX mapping analysis of C (\u003cem\u003ed\u003c/em\u003e), Fe (\u003cem\u003ee\u003c/em\u003e), N (\u003cem\u003ef\u003c/em\u003e), O (\u003cem\u003eg\u003c/em\u003e) elements of the sample; and, (\u003cem\u003eh\u003c/em\u003e) EDX spectra of these NPs.\u003c/p\u003e","description":"","filename":"Figure1MtezSoler.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4328624/v1/8f3b9bc019060ec3888b8dfc.jpg"},{"id":56275983,"identity":"67c7a7a7-c65b-497a-b812-0c1c760931dc","added_by":"auto","created_at":"2024-05-10 20:07:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":156494,"visible":true,"origin":"","legend":"\u003cp\u003eZeta potential (\u003cem\u003eζ\u003c/em\u003e, mV) of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e (■), CS (●), and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) (○) NPs as a function of: (\u003cem\u003ea\u003c/em\u003e) pH in the presence of 10\u003csup\u003e-3\u003c/sup\u003e M NaCl; and, (\u003cem\u003eb\u003c/em\u003e) the NaCl molar concentration at pH ≈ 4. Data is given as mean value ± SD (\u003cem\u003en\u003c/em\u003e = 9).\u003c/p\u003e","description":"","filename":"Figure2MtezSoler.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4328624/v1/a8519b62d3c6a4591da8c75b.jpg"},{"id":56275985,"identity":"afa1e22e-6e90-4669-885a-7c58df0d1863","added_by":"auto","created_at":"2024-05-10 20:07:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":286544,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cem\u003ea\u003c/em\u003e) Hysteresis cycle of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS particles; (\u003cem\u003eb\u003c/em\u003e) an aqueous dispersion of these NPs under the influence of a magnet placed close to the left lateral of the vial; optical microphotographs (magnification 20×) of the core/shell colloid (0.1%, w/v) under the influence of 0.4 T permanent magnet (in the direction of the arrow).\u003c/p\u003e","description":"","filename":"Figure3MtezSoler.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4328624/v1/5ecef53f3f27483e36593cc8.jpg"},{"id":56275943,"identity":"f5e315d8-6ea4-434d-819a-71c1074e4e36","added_by":"auto","created_at":"2024-05-10 20:07:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":134387,"visible":true,"origin":"","legend":"\u003cp\u003eRelease of Methotrexate (%) from the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) NPs as a function of the incubation time (h) at 37.0 ± 0.5 ºC and pH of the bloodstream (pH 7.4 ± 0.1, ●), or acidic environment in the endosomes and lysosomes of tumor cells (pH 5.5 ± 0.1, ○).\u003c/p\u003e","description":"","filename":"Figure4MtezSoler.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4328624/v1/99fda2c5c61e8c38962fcaaa.jpg"},{"id":56275987,"identity":"bfcb5297-39ce-4ffe-9755-c305fdeda436","added_by":"auto","created_at":"2024-05-10 20:07:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":183813,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cem\u003ea\u003c/em\u003e) Cytotoxicity of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) particles in MCF-10A breast epithelial cells and MCF-7 human breast cancer cells, after 72 h of exposure to a wide range of NP concentrations: 5 µM (grey column), 50 µM (light grey column), 100 µM (black column), and 200 µM (white column). (\u003cem\u003eb\u003c/em\u003e) Cytotoxicity of free MTX (white column), and MTX-loaded Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) NPs (light grey column) in MCF-7 human breast cancer cells, after 72 h of exposure to a wide range of NP concentrations (up to 100 µg/mL equivalent drug concentration). The values are the mean ± standard deviation (SD) (\u003cem\u003en\u003c/em\u003e = 4). Cells without treatment were used as control to calculate the relative cell viability (%). Inset: IC\u003csub\u003e50\u003c/sub\u003e values (µg/mL) of the MTX-based formulations.\u003c/p\u003e","description":"","filename":"Figure5MtezSoler.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4328624/v1/675c10751d5878747e4976c2.jpg"},{"id":64185640,"identity":"fb4f2f8c-4d60-43d7-b3a7-ee2f309357c0","added_by":"auto","created_at":"2024-09-09 16:18:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2159322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4328624/v1/369efa18-5cca-4bc5-bc1a-af2a310d3737.pdf"},{"id":56277147,"identity":"73fd0af2-4907-463c-98e8-8dfdafda578d","added_by":"auto","created_at":"2024-05-10 20:15:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":33520,"visible":true,"origin":"","legend":"","description":"","filename":"HighlightsMtezSoler.docx","url":"https://assets-eu.researchsquare.com/files/rs-4328624/v1/7a0e5a7cab942d46bacd131f.docx"},{"id":56275934,"identity":"99e7181d-0da8-4f3b-ba32-40ed0d06dc3a","added_by":"auto","created_at":"2024-05-10 20:07:05","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":199662,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstractMtezSoler.tif","url":"https://assets-eu.researchsquare.com/files/rs-4328624/v1/931c46e9e0ef99d1e645b705.tif"}],"financialInterests":"","formattedTitle":"Magnetic and pH-Responsive Magnetite/Chitosan (Core/Shell) Nanoparticles for Dual-Targeted Methotrexate Delivery in Cancer Therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMethotrexate (MTX), an analogue of folic acid, is a powerful chemotherapeutic agent and widely used for the treatment of human malignancies and autoimmune diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Its mechanism of action involves disrupting cellular folate metabolism by inhibiting dihydrofolate reductase, an essential enzyme for cell division. Despite its clinical efficacy, MTX faces challenges, including low specificity, dose-dependent undesirable side effects, the development of resistances in cancer cells and a potential risk of developing cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, MTX is a weak dicarboxylic acid [molecular weight, \u003cem\u003eM\u003c/em\u003e\u003csub\u003eW\u003c/sub\u003e: 54.44 g/mol (C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eN\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e); pKa values of 3.8, 4.8 and 5.6] that exhibit a poor, pH-dependent solubility (aqueous solubility\u0026thinsp;\u0026asymp;\u0026thinsp;0.01 mg/mL at 20\u0026deg;C) and low permeability\u003c/p\u003e \u003cp\u003e(C log\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;0.53). As a result, it has a poor oral bioavailability of less than 40%, which impairs its therapeutic effect, and therefore it is classified in class IV of the Biopharmaceutical Classification System [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, MTX is chemically unstable and, thus, undergoes rapid decomposition when exposed to light or subjected to extremes of pH or temperature. Consequently, it is often administered intravenously at high doses (\u0026ge;\u0026thinsp;500 mg/m\u003csup\u003e2\u003c/sup\u003e) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. To overcome these challenges, nanoparticles (NPs) have emerged as promising carriers for tailored MTX delivery [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNanotechnology has revolutionized cancer therapy by enabling targeted drug delivery, minimizing side effects and enhancing treatment efficacy [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Nanocarriers act as sophisticated vehicles, circulating through biological barriers to reach the specific cellular environments where their cargo can exert the most significant therapeutic effect [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Numerous research endeavors have delved into the encapsulation of MTX within NPs, with the primary objective of overcoming drug resistances in cancer cells and enhancing therapeutic outcomes. The intricately tailored encapsulation within NPs aims to provide sustained release profiles, ensuring a prolonged and controlled delivery of MTX. This sustained release approach plays a pivotal role in circumventing the development of resistances. Furthermore, nanoformulations contribute to improved bioavailability, enhancing the amount of MTX reaching its target within the body, thereby maximizing its therapeutic impact [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous polymers have recently been suggested to formulate MTX-loaded NPs for cancer therapy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Among these polymers, chitosan (CS), a naturally derived polysaccharide from chitin, offers a wealth of advantages that render it an ideal candidate for use in the design of nanoparticulate drug delivery systems. Known for its hydrophilicity, biocompatibility, low immunogenicity, biodegradability and mucoadhesive properties, CS has found widespread application in diverse medical formulations [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. CS\u0026rsquo;s appeal in the medical field extends beyond its biological properties to include its ease of modification and versatility. The presence of reactive amino and hydroxyl groups allows for various chemical modifications, facilitating the tailoring of CS-based formulations to meet specific medical requirements [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, the cationic nature of chitosan enables interactions with negatively charged biological surfaces, a feature strategically employed in NP development [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. When employed as a coating polymer for NPs, chitosan not only imparts stability and controlled release but also introduces an additional layer of biocompatibility, offering a hydrophilic stealth coating that prevent opsonization and evade phagocytosis in mononuclear phagocyte system, which is crucial for navigating the blood circulation effectively [\u003cspan additionalcitationids=\"CR40 CR41 CR42 CR43 CR44\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral MTX-loaded CS-based NPs have been designed to enhance controlled drug delivery to tumors [\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. These include actively targeted NPs, such as those using covalently conjugated to folate [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], as well as stimuli-responsive particles. Stimuli-responsive nanosystems encompass pH-responsive NPs, for instance, by incorporating the surfactant 77 KL (\u003cem\u003eN\u003c/em\u003e\u003csup\u003eα\u003c/sup\u003e,\u003cem\u003eN\u003c/em\u003e\u003csup\u003eε\u003c/sup\u003e-dioctanoyl lysine with a lithium counterion) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], redox-responsive NPs, like those based on \u003cem\u003eL\u003c/em\u003e-cysteine covalently linked to CS [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and light-responsive NPs, functionalized with photocatalytic titanium dioxide NPs [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These innovative formulations, often utilizing modified forms of CS, aim to enhance the targeting capability of MTX-loaded CS NPs and exert increased control over the release of the chemotherapeutic agent. While many studies showcase the improved efficiency of MTX-loaded CS-based NPs, a notable limitation is the lack of comprehensive preclinical data in several of these investigations. Future research should aim to bridge this gap, providing a more robust foundation for the translation of these findings into clinical applications.\u003c/p\u003e \u003cp\u003eFurthermore, magnetically driven NPs can be advantageous in achieving targeted delivery to tumor cells and other disorders [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Magnetite (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) stands out as a distinctive category of magnetic nanoparticles that has garnered considerable attention the medical field. Due to their biocompatibility, extensive chemical affinity and distinctive magnetic properties, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs have found widespread use in biomedical applications [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. A primary application within the medical domain is evident in magnetic resonance imaging, where the superparamagnetic characteristics of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e contribute to enhanced imaging contrast, facilitating clearer and more detailed visualization of tissues and organs. In addition, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e particles play a crucial role in targeted drug delivery systems, enabling the precise attachment or encapsulation of drugs within these NPs for specific delivery to targeted cells or tissues [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. This targeted strategy not only enhances the therapeutic effectiveness of drugs but also minimizes potential side effects. Despite their immense potential, the use of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs in medical applications presents certain challenges. One notable hurdle involves concerns about potential toxicity, as the body\u0026rsquo;s response to iron-based NPs needs careful consideration. Another obstacle pertains to the susceptibility of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e to air oxidation, which can lead to the loss of magnetic properties. Maintaining the stability of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e particles in physiological environments and preventing aggregation are critical for successful medical applications. In addressing these challenges, researchers have directed their efforts toward surface modifications of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs. Coating these particles with various materials, including natural substances such as CS or synthetic polymers like polyethylene glycol and polyvinylpyrrolidone, enhances their biocompatibility and stability. Furthermore, endeavors have been undertaken to optimize the size, shape, surface charge and functionalization of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs, aiming to enhance their overall performance in Biomedicine [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e is incorporated within the CS matrix the resulting core/shell nanocomposite combines the magnetic responsiveness of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs with the inherent benefits of CS [\u003cspan additionalcitationids=\"CR55 CR56\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. These magnetically driven NPs have shown promise in targeted drug delivery, hyperthermia, and imaging applications. The magnetic responsiveness enables the NPs to be guided to specific sites within the body using external magnetic fields. This targeted approach enhances the precision of drug delivery, minimizing off-target effects and improving therapeutic outcomes. Furthermore, the combination of CS and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e in NPs offers opportunities for multifunctionality. Beyond drug delivery, these particles can be explored for applications such as magnetic hyperthermia, where the magnetic properties are harnessed to generate localized heat for therapeutic purposes [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we explore magnetically targeted and pH-responsive Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) nanocomposites as a drug delivery platform for MTX. The research delves into the formulation and characterization of these NPs, emphasizing their stability, MTX loading capacity and magnetic responsiveness. In addition, the potential pH-responsive MTX release behavior is investigated under the acidic microenvironment typical of tumors (pH\u0026thinsp;\u0026asymp;\u0026thinsp;5.5). Furthermore, the hemocompatibility and cytotoxicity of these MTX-loaded magnetic nanocomposites against human breast cancer cells are thoroughly examined, providing crucial insights into their safety and efficacy profiles. To the best of our knowledge, this is the first time that these Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) NPs with dual targeted features, magnetic and pH responsiveness, have been developed as a platform for the delivery of MTX molecules to malignant cells. By combining the advantageous features of the developed NPs, this research aims to significantly contribute to the advancement of targeted MTX-based cancer therapy.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. Materials\u003c/h2\u003e\n \u003cp\u003eAll chemicals were of analytical quality. Iron(III) chloride hexahydrate, iron(II) chloride tetrahydrate, hydrochloric acid, perchloric acid, and acetic acid were purchased from VWR International, LLC (Spain). Kolliphor\u0026reg; P-188 was purchased from BASF (Germany). CS (M\u003csub\u003eW\u003c/sub\u003e \u0026asymp; 50 to 190 kDa, 75\u0026ndash;85% deacetylated), ammonia, citric acid (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e), sodium sulfate, sodium citrate, sodium hydroxide (NaOH), sodium chloride (NaCl), disodium hydrogen phosphate (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e), ethylenediaminetetraacetic acid (EDTA), phosphate buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl tetrazolium bromide (MTT), MTX, dimethyl sulfoxide, Dulbecco\u0026rsquo;s modified eagle\u0026rsquo;s medium (DMEM), fetal bovine serum (FBS), and Penicillin-Streptomycin solution (containing 10,000 U/mL of Penicillin and 10 mg/mL of Streptomycin) were purchased from Merck KGaA (Germany). Water was deionized and filtered with a Milli-Q Academic\u0026reg; system (Millipore, Spain).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. Methods\u003c/h2\u003e\n \u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003e2.2.1. Formulation of magnetite, chitosan, and magnetite/chitosan (core/shell) nanoparticles\u003c/h2\u003e\n \u003cp\u003eFe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were prepared using a controlled chemical co-precipitation process [52\u0026ndash;55]. Initially, 40 mL of 2 M iron(III) chloride solution and 10 mL of 1 M iron(II) chloride solution (in 2 M hydrochloric acid) were gradually added to 500 mL of 1.5 M ammonia solution, under mechanical stirring (700 rpm; IKA\u0026reg; Eurostar 60 Digital Constant-Speed Mixer, Germany) at room temperature. After 30 min of continuous stirring, the resulting iron oxide particles were magnetically separated with a 0.4 T permanent magnet and re-dispersed in a 2 M nitric acid solution to achieve a stable aqueous dispersion. Following 1 h, the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were magnetically decanted again, followed by systematic cleaning through repeated magnetic separation and redispersion in water until the supernatant became transparent, and its conductivity indicated the absence of both unreacted chemicals and non-magnetic particles (achieved conductivity of the supernatant was \u0026le;\u0026thinsp;10 \u0026micro;S\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Crison Microcm 2202 conductivity meter, Hach Lange Spain S.L.U., Spain). Subsequently, the NPs were dried at 60.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C in a convection oven (J.P. Selecta, S.A., Spain) and stored for later use.\u003c/p\u003e\n \u003cp\u003eCS NPs were produced using a coacervation method, wherein the introduction of sodium sulfate into a solution of CS in acetic acid reduces the solubility of the polymer, leading to its rapid precipitation into NPs [38\u0026ndash;40, 52, 54]. Specifically, a 1% (w/v) solution of CS was prepared in 50 mL of an aqueous solution of acetic acid (2%, v/v) containing 1% (w/v) Kolliphor\u0026reg; P-188, and the pH was adjusted to pH 4 with 1 M sodium hydroxide solution. Subsequently, 12.5 mL of a sodium sulfate solution (20%, w/v) was added drop-wise (at a rate of 2.5 mL/min) to the CS solution while mechanically stirring at 1,200 rpm. The stirring continued for 1 h to yield an aqueous dispersion of CS NPs. To purify the colloid, a cleaning process involving multiple cycles of centrifugation at 11,000 rpm for 60 min (Centrifuge 5804, Eppendorf Ib\u0026eacute;rica S.L.U., Spain) and redispersion in water was followed. This purification process was repeated until the conductivity of the supernatant reached\u0026thinsp;\u0026le;\u0026thinsp;10 mS\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eMagnetically responsive Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) NPs were produced using a coacervation method identical to the one employed for the CS particles described above. In the initial step of the formulation process, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nuclei (0.75%, w/v) were introduced into the aqueous acetic acid solution used for CS dissolution. Subsequently, the NP dispersion was subjected to a magnetic purification procedure, where the resulting NPs were repetitively isolated from the liquid medium, using a 0.4 T permanent magnet, and redispersed in pure water until the supernatant\u0026rsquo;s conductivity reached\u0026thinsp;\u0026le;\u0026thinsp;10 mS\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eMTX was loaded to the magnetic core/shell NPs using an entrapment technique. The same procedure used for obtaining the CS-coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs was followed, where the anticancer agent was dissolved in the aqueous polymer solution of pH\u0026thinsp;\u0026asymp;\u0026thinsp;4. After the preparation process, any excess of drug molecules was eliminated through magnetic cleaning, as described above. To study the effect of MTX concentration on its loading, drug concentrations ranging from 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M were examined.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e2.2.2. Characterization of size, surface electrical charge, and short-term stability\u003c/h2\u003e\n \u003cp\u003eThe mean particle size and size distribution (polydispersity index, PdI) as well as the zeta potential (\u003cem\u003e\u0026zeta;\u003c/em\u003e) of the NPs were determined by photon correlation spectroscopy and electrokinetic determinations, respectively, after appropriate dilution of the aqueous colloids (\u0026asymp;\u0026thinsp;0.1%, w/v). These measurements were performed using a Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK). The experiments were conducted at a constant cell temperature of 25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, and the detection angle employed was 60\u0026deg;.\u003c/p\u003e\n \u003cp\u003eFurthermore, size and surface characteristics of the magnetic particles were examined using high resolution transmission electron microscopy (HRTEM), annular bright field scanning transmission electron microscopy (ABF-STEM) and high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) (Titan G2 60\u0026ndash;300 FEI microscope, Thermofisher Scientific Inc., USA; operating at an accelerating voltage of 300 kV). Prior to observation, diluted aqueous NP dispersions (\u0026asymp;\u0026thinsp;0.1%, w/v) were sonicated for 5 min, and droplets were placed on formvar/carbon-coated copper microgrids. Subsequently, the samples were dried in a convection oven (25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C) (J. P. Selecta, S. A., Spain). Elemental analysis was conducted simultaneously with the TEM measurements using an energy dispersive X-ray (EDX) spectrometer (Bruker Nano GmbH, Germany).\u003c/p\u003e\n \u003cp\u003eAn assessment of the CS coating on Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs was conducted by analyzing the influence of pH and ionic strength on the \u003cem\u003e\u0026zeta;\u003c/em\u003e of the particles [39, 40, 58]. To assess the effect of pH, the study encompassed a range from pH 3 to 9, in the presence of 1 mM NaCl. Additionally, the impact of ionic strength was examined using various concentrations of NaCl while maintaining a constant pH\u0026thinsp;\u0026asymp;\u0026thinsp;4. These determinations were carried out at room temperature (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9) after 24 h of contact under stirring conditions (200 rpm, Boeco universal orbital shaker OS-10, Boeco, Germany).\u003c/p\u003e\n \u003cp\u003eMoreover, the short-term stability of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) colloidal formulation (1 mg/mL, pH\u0026thinsp;\u0026asymp;\u0026thinsp;6) was evaluated through incubation at 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C and 25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C for a duration of 90 days. Throughout the experiment, the size, PdI and \u003cem\u003e\u0026zeta;\u003c/em\u003e values of the NPs were measured to monitor any changes in their properties.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e2.2.3. Exploring magnetic properties\u003c/h2\u003e\n \u003cp\u003eThe magnetic characteristics of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) particles were investigated using a Quantum Design MPMS XL (USA) SQUID magnetometer at room temperature. In addition, the magnetic responsiveness of the NPs was qualitatively assessed by observing a 0.1% (w/v) aqueous dispersion under a 0.4 T permanent magnet, using a Nikon SMZ800 stereoscopic zoom microscope (Nikon, Japan) [38, 59, 60].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003e2.2.4. Quantification of Methotrexate loading and in vitro release\u003c/h2\u003e\n \u003cp\u003eThe determination of the amount of drug loaded to the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/chitosan NPs was carried out by UV spectrophotometric analyses of the MTX remaining in the supernatant after NP centrifugation (60 min at 11,000 rpm). The loaded drug quantity was calculated by subtracting the drug released into the medium from the total amount used during particle preparation. To account for possible absorbance contributions of other formulation components, e.g. the surfactant agent, the absorbance of the supernatant from blank NPs (containing no MTX) was subtracted. MTX concentration was measured at its maximum absorbance wavelength (\u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;304 nm) by Ultraviolet\u0026ndash;Visible spectroscopy (UV\u0026ndash;Vis Dinko spectrophotometer, Dinko, Spain). Finally, the drug content was expressed as entrapment efficiency (EE, %) and drug loading (DL, %) according to Equations 1 and 2.\u003c/p\u003e\n \u003cdiv id=\"Equ1\"\u003e\n \u003cdiv id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$EE \\left(\\text{%}\\right)=\\frac{\\text{t}\\text{o}\\text{t}\\text{a}\\text{l} \\text{M}\\text{T}\\text{X} \\text{a}\\text{m}\\text{o}\\text{u}\\text{n}\\text{t} \\left(\\text{m}\\text{g}\\right)-\\text{u}\\text{n}\\text{e}\\text{n}\\text{c}\\text{a}\\text{p}\\text{s}\\text{u}\\text{l}\\text{a}\\text{t}\\text{e}\\text{d} \\text{M}\\text{T}\\text{X} \\text{a}\\text{m}\\text{o}\\text{u}\\text{n}\\text{t} \\left(\\text{m}\\text{g}\\right)}{\\text{t}\\text{o}\\text{t}\\text{a}\\text{l} \\text{M}\\text{T}\\text{X} \\text{a}\\text{m}\\text{o}\\text{u}\\text{n}\\text{t} \\left(\\text{m}\\text{g}\\right)} \\times 100$$\u003c/div\u003e\u003cdiv\u003e1\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ2\"\u003e\u003cdiv id=\"FileID_Equ2\" name=\"EquationSource\"\u003e$$DL \\left(\\text{%}\\right)= \\frac{\\text{t}\\text{o}\\text{t}\\text{a}\\text{l} \\text{M}\\text{T}\\text{X} \\text{a}\\text{m}\\text{o}\\text{u}\\text{n}\\text{t} \\left(\\text{m}\\text{g}\\right)-\\text{u}\\text{n}\\text{e}\\text{n}\\text{c}\\text{a}\\text{p}\\text{s}\\text{u}\\text{l}\\text{a}\\text{t}\\text{e}\\text{d} \\text{M}\\text{T}\\text{X} \\text{a}\\text{m}\\text{o}\\text{u}\\text{n}\\text{t} \\left(\\text{m}\\text{g}\\right)}{\\text{t}\\text{o}\\text{t}\\text{a}\\text{l} \\text{m}\\text{a}\\text{s}\\text{s} \\text{o}\\text{f} \\text{N}\\text{P}\\text{s} \\left(\\text{m}\\text{g}\\right)} \\times 100$$\u003c/div\u003e\n \u003cdiv\u003e2\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eDrug release from the CS-coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e particles were studied in buffers at two distinct pH values: a C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e-NaOH buffer at pH 5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 (pH of acidic microenvironment in tumors) and a C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e-Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e buffer at pH 7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 (pH of bloodstream). The magnetic colloid, 2 mL of NPs containing 5 mg/mL of MTX, was dispersed in 200 mL of each buffer. Throughout the experiment, dispersions were maintained at 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C and under mechanical stirring (100 rpm). At predetermined time intervals, 1 mL of the release medium was withdrawn and the concentration of MTX was determined by UV\u0026ndash;Vis spectroscopy at 304 nm. After each sampling, an equal volume of the release medium, maintained at 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, was replenished to maintain the volume of the release medium and ensure sink conditions. The same analytical procedure used to determine the DL (%) was applied, and the \u003cem\u003ein vitro\u003c/em\u003e MTX release was calculated using Eq.\u0026nbsp;3:\u003c/p\u003e\n \u003cdiv id=\"Equ3\"\u003e\n \u003cdiv id=\"FileID_Equ3\" name=\"EquationSource\"\u003e$$\\text{C}\\text{u}\\text{m}\\text{u}\\text{l}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e} \\text{M}\\text{T}\\text{X} \\text{r}\\text{e}\\text{l}\\text{e}\\text{a}\\text{s}\\text{e} \\left(\\text{%}\\right)= \\frac{\\text{a}\\text{m}\\text{o}\\text{u}\\text{n}\\text{t} \\text{o}\\text{f} \\text{M}\\text{T}\\text{X} \\text{r}\\text{e}\\text{l}\\text{e}\\text{a}\\text{s}\\text{e}\\text{d} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{m}\\text{e}\\text{d}\\text{i}\\text{u}\\text{m} \\left(\\text{m}\\text{g}\\right) }{\\text{a}\\text{m}\\text{o}\\text{u}\\text{n}\\text{t} \\text{o}\\text{f} \\text{M}\\text{T}\\text{X} \\text{l}\\text{o}\\text{a}\\text{d}\\text{e}\\text{d} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{N}\\text{P}\\text{s} \\left(\\text{m}\\text{g}\\right)} \\times 100$$\u003c/div\u003e\u003cdiv\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\"\u003e\u003ch2\u003e2.2.5. Ex vivo hemocompatibility\u003c/h2\u003e\u003cdiv\u003e\u003cp\u003eThe evaluation of the nanosystem\u0026rsquo;s hemocompatibility provides valuable insights into its potential clinical applications. Human blood, sourced from healthy donors, was collected into flasks containing EDTA (for hemolysis and platelet activation experiments) or sodium citrate (for complement system activation and plasma clotting time assays), and treated following established procedures [15, 61, 62]. PBS served as the negative control in these experiments. The developed particles were incubated during 24 h with blood aliquots to assess their impact on erythrocyte lysis (hemoglobin release), complement activation (C3a release), platelet activation (sP-selectin release), and plasma recalcification time (T\u003csub\u003e1/2\u003c/sub\u003e max), employing validated UV\u0026ndash;Vis spectrophotometric methodologies.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\"\u003e\u003ch2\u003e2.2.6. In vitro cell culture experiments\u003c/h2\u003e\u003cdiv id=\"Sec11\"\u003e\u003ch2\u003e2.2.6.1. Cell maintenance\u003c/h2\u003e\u003cdiv\u003e\u003cp\u003eMCF-10A human breast epithelial cells and MCF-7 human breast cancer cells (American Type Culture Collection, USA) were cultured in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin solution, and were maintained in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C until used in the experiments [MCO-19AIC(UV) CO\u003csub\u003e2\u003c/sub\u003e incubator, Sanyo, Japan].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\"\u003e\u003ch2\u003e2.2.6.2. In vitro cytotoxicity assay\u003c/h2\u003e\u003cdiv\u003e\u003cp\u003eThe cytotoxicity of MTX-loaded NPs was assessed using the MTT proliferation assay, which determines mitochondrial dehydrogenase activity. Various concentrations of free MTX, blank NPs and MTX-loaded NPs, dissolved or dispersed in the culture medium, were added to the cells and incubated for 72 h at 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. Subsequently, MTT solution (20 \u0026micro;L/well, 5 mg/mL in cell culture medium) was added and the cells were further incubated for 3 h at 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C. After removal of the culture medium, formazan crystals were dissolved in 200 \u0026micro;L of dimethyl sulfoxide. The optical density (OD) of the resulting dye, proportional to the number of viable (metabolically active) cells, was measured at 570 nm (Dynatech MR7000 microplate reader, Dynatech Laboratories, Inc., USA). Cells treated with Triton\u003csup\u003e\u0026reg;\u003c/sup\u003e X-100 (1%, v/v) or incubated with cell culture medium without treatment served as controls. The relative cell viability (RCV, %) was calculated using Eq.\u0026nbsp;4.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Equ4\"\u003e\u003cdiv id=\"FileID_Equ4\" name=\"EquationSource\"\u003e$$\\text{R}\\text{C}\\text{V} \\left(\\text{%}\\right)= \\frac{\\text{O}\\text{D} \\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{e}\\text{d} \\text{c}\\text{e}\\text{l}\\text{l}\\text{s} }{\\text{O}\\text{D} \\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l} \\left(\\text{u}\\text{n}\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{e}\\text{d}\\right) \\text{c}\\text{e}\\text{l}\\text{l}\\text{s}} \\times 100$$\u003c/div\u003e\n \u003cdiv\u003e4\u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values were calculated through non-linear regression analysis using GraphPad Prism 9.1.0 software (GraphPad Software Inc., USA).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e2.2.7. Statistical analysis\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe collected data were subjected to statistical analysis using the SPSS Statistics software package (version 26.0; IBM Corporation, USA). Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was utilized for comparing results, ensuring a 95% confidence interval. Each experiment was conducted in three independent assays. The results were presented as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance was defined at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Size, surface electrical charge, and short-term stability\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe characteristics of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e /CS (core/shell) NPs are demonstrated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (time 0 days). While the mean diameter of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e particles was \u0026asymp;\u0026thinsp;11 nm, it was remarkably increased when these iron oxide nuclei were surface coated with CS to \u0026asymp;\u0026thinsp;270 nm. In addition, the PdI also increased from \u0026asymp;\u0026thinsp;0.031 to \u0026asymp;\u0026thinsp;0.138 for unmodified and coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean diameter (nm), PdI, and \u003cem\u003eζ\u003c/em\u003e data (mV\u003cem\u003e)\u003c/em\u003e of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) NPs as a function of time (days) at 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C or 25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C. Experimental values are indicated as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eShort-term stability assay at 25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c8\" namest=\"c5\"\u003e \u003cp\u003eShort-term stability assay at 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSize (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePdI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eζ\u003c/em\u003e (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTime (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSize (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePdI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eζ\u003c/em\u003e (mV)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e270.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.138\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e270.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.138\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e263.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.142\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e25.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e279.6\u0026thinsp;\u0026plusmn;\u0026thinsp;11.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.161\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e24.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e287.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.126\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e260.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.147\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e28.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e282.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.131\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e22.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e289.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.107\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e24.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e273.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.141\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e294.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.129\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e23.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e60\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e313.9\u0026thinsp;\u0026plusmn;\u0026thinsp;11.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.163\u0026thinsp;\u0026plusmn;\u0026thinsp;0.038\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e19.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e60\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e339.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.157\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e19.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e90\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e372.2\u0026thinsp;\u0026plusmn;\u0026thinsp;23.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.191\u0026thinsp;\u0026plusmn;\u0026thinsp;0.033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e90\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e391.3\u0026thinsp;\u0026plusmn;\u0026thinsp;29.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.203\u0026thinsp;\u0026plusmn;\u0026thinsp;0.043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe core/shell structure and elemental composition of the CS-coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were examined through electron microscopy and EDX mapping analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The HRTEM image (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), alongside HAADF-STEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and ABF-STEM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) clearly illustrated the embedding of iron oxide nuclei within the polymeric matrices. The observed clustering of NPs was likely a consequence of the sample preparation method involving drying for microscopy observations, a phenomenon documented in existing literature [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. EDX analyses further validated the NP composition, verifying the presence of Fe, C, N and O elements within the NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Specifically, the Fe and O elements were associated with the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e cores, while the N and C elements originated from the CS shell. The detection of Cu and Si elements in the EDX analysis could be explained by the utilization of copper-based grids [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] and the secondary fluorescence generated and detected by the fluorescence detector [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Elemental mapping of Fe and O (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg) highlighted the uniform distribution of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nuclei within the NP matrix, while the mapping of C and N (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef) demonstrated the effective coating of CS onto the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanostructures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the coating efficiency of CS around the iron oxide particles, electrophoretic characteristics of the colloids were studied (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At pH 3, all NPs, including Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, CS and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS NPs, displayed a positive \u003cem\u003eζ\u003c/em\u003e ranging from +\u0026thinsp;50 to +\u0026thinsp;60. With increasing pH, both CS and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS NPs showed a gradual decrease in \u003cem\u003eζ\u003c/em\u003e, approaching electroneutrality at pH 9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Conversely, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e particles exhibited a steeper decline, reaching the isoelectric point, or pH of zero \u003cem\u003eζ\u003c/em\u003e, at pH\u0026thinsp;\u0026asymp;\u0026thinsp;7, and dramatically dropping to \u0026asymp; -90 at pH 9. Furthermore, when the NaCl concentration (at pH\u0026thinsp;\u0026asymp;\u0026thinsp;4) was raised from 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M to 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e M, the \u003cem\u003eζ\u003c/em\u003e of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS NPs increased from \u0026asymp;\u0026thinsp;+\u0026thinsp;30 to \u0026asymp;\u0026thinsp;+\u0026thinsp;40, similar to the trend observed with CS NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In contrast, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs showed minimal variation in the \u003cem\u003eζ\u003c/em\u003e across the tested range of ionic strengths.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, short-term stability of aqueous dispersion of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) NPs at both 4 \u0026ordm;C and 25 \u0026ordm;C demonstrated relatively stable particle size and surface electrical charge (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The NPs showed high stability under the used storage conditions, both 4 \u0026ordm;C and 25 \u0026ordm;C, over the first 30 days as can be observed by the minimal changes in their characteristics. However, when the particles were further stored up to 90-day, they were found to grow in size by \u0026asymp;\u0026thinsp;1.4-fold, under both storage conditions. A similar trend was also observed with the PdI values which increased from \u0026asymp;\u0026thinsp;0.14 to \u0026asymp;\u0026thinsp;0.19.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Magnetic responsiveness\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe magnetic responsiveness of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS NPs was investigated by analyzing the hysteresis cycle, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. The Figure clearly illustrates the nanocomposite\u0026rsquo;s soft magnetic character, as the increasing and decreasing field branches of the hysteresis cycle are hardly distinguishable. By examining the linear portions (low field) of the curve, it was possible to estimate the initial susceptibility as (0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03) \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e m\u003csup\u003e3\u003c/sup\u003e/Kg, and the saturation magnetization as 19.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06 Am\u003csup\u003e2\u003c/sup\u003e/Kg for the NPs. The effective magnetic response of the core/shell particles was further qualitatively confirmed by observing the colloid\u0026rsquo;s behavior when exposed to a permanent magnet (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Notably, the NPs exhibited complete magnetic attraction towards a 0.4 T magnet within 45 s. Additionally, the assessment of the nanocomposite\u0026rsquo;s magnetic responsiveness was extended through optical microscope visualization of the colloid under the influence of the magnet (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Initially, the aqueous dispersion of particles was uniformly distributed. However, upon exposure to the magnetic field, significant changes occurred. Chainlike aggregates formed parallel to the field lines, providing visual evidence of the NPs\u0026rsquo; magnetic responsiveness.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Methotrexate loading and release\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe EE (%) and DL (%) of MTX in the CS-coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e particles are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. With an increase in drug concentration during NP preparation, ranging from 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M, the drug loading capacity experienced significant increments. Specifically, EE (%) and DL (%) values increased substantially from \u0026asymp;\u0026thinsp;5% and \u0026asymp;\u0026thinsp;0.009% to \u0026asymp;\u0026thinsp;36% and \u0026asymp;\u0026thinsp;0.65%, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLoading of Methotrexate (EE and DL, %) to the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) particles. Experimental values are indicated as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e[Methotrexate] (M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEE (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDL (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.009\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.082\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.199\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e29.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.374\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e35.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.651\u0026thinsp;\u0026plusmn;\u0026thinsp;0.039\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe pH-responsive MTX release from the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) NPs was assessed at 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C by using release media stimulating the pH conditions of the bloodstream and the acidic environment in the endosomes and lysosomes of tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At pH\u0026thinsp;\u0026asymp;\u0026thinsp;7.4, a distinct biphasic drug release profile was observed, characterized by an initial rapid burst release of \u0026asymp;\u0026thinsp;60% of MTX within the first 6 h. Subsequently, the remaining chemotherapeutic was released at a slower rate over the next 90 h. In contrast, at pH\u0026thinsp;\u0026asymp;\u0026thinsp;5.5, a significantly faster drug release rate (\u0026asymp;\u0026thinsp;2.9-fold) was noted, leading to complete MTX release achieved within only 3 h (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Ex vivo hemocompatibility\u003c/h2\u003e \u003cp\u003eThe blood compatibility tests conducted on blank core/shell particles are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. These magnetic nanocomposites exhibited minimal erythrolytic activity even after 24 h of incubation, with only\u0026thinsp;\u0026asymp;\u0026thinsp;2% hemolysis observed. Furthermore, the analysis of complement system activation, platelet activation and plasma clotting times indicated that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS NPs had no significant impact on related parameters such as sP-selectin release, C3a release and T\u003csub\u003e1/2 max\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) NPs on hemolysis (%), complement activation (C3a release: C3a desArg, ng/mL), platelet activation (sP-selectin release, ng/mL), and plasma recalcification time (T\u003csub\u003e1/2 max\u003c/sub\u003e, min). Data is expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS NPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl (PBS solution)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003eHemolysis (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncubation time: 2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncubation time: 6 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncubation time: 12 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncubation time: 24 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC3a desArg (ng/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e299.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e301.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003esP-selectin release (ng/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e101.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e102.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003e1/2\u003c/b\u003e \u003cb\u003emax\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(min)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. In vitro cytotoxicity assay\u003c/h2\u003e \u003cp\u003eThe results of the cytotoxic evaluation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS particles in MCF-10A breast epithelial cells and MCF-7 human breast cancer cells after 72 h of incubation are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. It is evident that increasing concentrations of the blank core/shell NPs (from 5 to 200 \u0026micro;M) induced minimal cytotoxicity in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb demonstrates a dose-dependent inhibition of cancer cell growth observed with MTX treatment, both free and loaded to the NPs. In comparison with free drug, MTX-loaded magnetic particles significantly enhanced the MTX antitumor activity at concentrations ranging from 10 to 100 \u0026micro;g/mL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, the IC\u003csub\u003e50\u003c/sub\u003e of the MTX-loaded core/shell NPs (26.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 \u0026micro;g/mL) was \u0026asymp;\u0026thinsp;2.7-fold less than that of the free anticancer drug (72.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 \u0026micro;g/mL) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn this study, magnetically responsive Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS particles loaded with the chemotherapeutic MTX were developed and extensively characterized. The formulated NPs demonstrated good loading capacity for the anticancer therapeutic agent and promising characteristics for possible biomedical applications. Thus, their potential as a targeted drug delivery system for cancer treatment was explored using various \u003cem\u003ein vitro\u003c/em\u003e experiments.\u003c/p\u003e \u003cp\u003eBuilding on previous research, the preparation of the core/shell NPs was convincingly demonstrated through the effective and uniform embedding of iron oxide nuclei within the CS polymeric coating. This was confirmed through the morphological analysis under the electron microscope and elemental mappings via EDX. The formulation method involved initially obtaining Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e particles, followed by chitosan coacervation around the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nuclei, a widely employed procedure for creating CS-coated magnetic NPs [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Although CS surface functionalization of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs is associated with an increase in size (from \u0026asymp;\u0026thinsp;11 nm to \u0026asymp;\u0026thinsp;270 nm), it imparts a hydrophilic positive surface charge that may protect iron oxide particles from capture by the mononuclear phagocytic system, thus probably preventing rapid clearance from the systemic circulation and making the nanocomposites suitable for parenteral administration. Consequently, these stealth properties, combined with the appropriate size range, may enhance the NPs\u0026rsquo; plasma half-lives, facilitating their delivery to the tumor site and extravasation into tissues through the gaps between endothelial cells of the tumor vasculature (up to \u0026asymp;\u0026thinsp;600 nm), capitalizing on the enhanced permeability and retention effect [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Moreover, the positive surface charge conferred by CS prevents particle aggregation, thanks to the electrostatic repulsion generated. This, in turn, ensures the physical stability of the NPs when in aqueous dispersion. Notably, short-term stability analysis conducted in this study confirmed the efficacy of the preparation method in formulating well-stabilized core/shell NPs. In fact, the nanocomposites maintained a stable size and surface electrical charge, with no observable aggregation for at least 30 days when stored at both 4 \u0026ordm;C and 25 \u0026ordm;C (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings were also confirmed when the electrophoretic properties of the NPs were examined as a function of pH and ionic strength. CS-coated NPs exhibited a positive \u003cem\u003eζ\u003c/em\u003e between +\u0026thinsp;40 and +\u0026thinsp;50 at pH\u0026thinsp;\u0026asymp;\u0026thinsp;5, indicating their stability and potential for controlled release under acidic conditions, such as in the tumor microenvironment. The behavior of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS NPs in the presence of varying ionic strengths further highlighted their stability and suitability for biological applications. In biological settings, positively charged NPs exhibit an ability to interact with negatively charged mucus and cells, leading to improved permeation, absorption and bioavailability of the particles and their cargo. In addition, the mucoadhesive characteristics exhibited by CS-coated NPs may play a role in prolonging the drug\u0026rsquo;s action by extending their residence time at mucous-rich sites. Moreover, these positive surface electrical charges could potentially enhance the internalization of NPs by negatively charged cancer cells, ensuring the delivery of the therapeutic molecule to the intracellular targets [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, the incorporation of MTX, which is a poly-functional weak dicarboxylic acid (pKa values of 3.8, 4.8 and 5.6) [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], into the CS matrix could be the consequence of electrostatic attractions between drug molecules, negatively charged when the \u0026ndash;COOH groups are protonated, and the positively charged polymer (\u0026thinsp;\u0026asymp;\u0026thinsp;+\u0026thinsp;40 mV at pH 4). The electrostatic interaction between MTX and CS also influences the drug release profile resulting in a pH-responsive MTX release behavior. While under pH\u0026thinsp;\u0026asymp;\u0026thinsp;7.4, MTX shows a sustained release over the next 90 h, the chemotherapeutic is completely released within 3 h under acidic conditions (pH 5.5). The observed pH-responsiveness, as previously documented in studies involving CS-coated NPs [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], may be attributed to the presence of the hydrophilic CS coating. With a pKa of 6.5, the amine groups of CS undergo ionization in acidic solutions, making it more soluble at lower pH levels. This, in turn, accelerates drug diffusion and release [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. This behavior aligns with the targeted drug delivery requirements, ensuring preferential drug release in the acidic tumor microenvironment, thereby enhancing therapeutic efficacy while minimizing systemic side effects.\u003c/p\u003e \u003cp\u003eThe magnetic responsiveness of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS NPs was demonstrated through hysteresis cycle analysis and visual observations under the influence of a magnetic field. The soft magnetic nature of the nanocomposites was evident, and their rapid response to a magnetic field, forming chainlike aggregates parallel to the field lines, indicated their potential for targeted drug delivery [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. This could result from the notable contribution of the magnetic interaction over the Derjaguin-Landau-Verwey-Overbeek (DLVO) colloidal interactions between the NPs (\u003cem\u003ee.g.\u003c/em\u003e electrostatic van der Waals and hydration or acid\u0026ndash;base) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. However, \u003cem\u003ein vivo\u003c/em\u003e experiments should be done to clarify if this magnetic responsiveness could determine the accumulation of the CS-coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs at a targeted site.\u003c/p\u003e \u003cp\u003eIn the \u003cem\u003eex vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e biocompatibility assessments, the core/shell particles exhibited excellent hemocompatibility, and no significant impact on blood components. In addition, these NPs showed no \u003cem\u003ein vitro\u003c/em\u003e cytotoxic effects on both normal and cancer cells. These results underscore the biocompatibility and safety of the nanocomposites, suggesting their potential for intravenous administration without inducing significant adverse effects.\u003c/p\u003e \u003cp\u003eLastly, MTX-loaded NPs exhibited a dose-dependent inhibition of cancer cell growth, significantly enhancing the antitumor activity compared to free MTX. The substantially lower IC\u003csub\u003e50\u003c/sub\u003e of the MTX-loaded NPs compared to the free drug, down by \u0026asymp;\u0026thinsp;2.7-fold, indicated a significant enhancement in the therapeutic efficacy of the formulated particles. These findings support the potential of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS NPs as a promising platform for targeted delivery of MTX in cancer therapy, warranting further investigations into their \u003cem\u003ein vivo\u003c/em\u003e safety and efficacy.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn this research, magnetically targeted MTX-loaded Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CS (core/shell) NPs were developed and thoroughly characterized. These particles exhibited favorable characteristics, including effective CS coating, stability, magnetic responsiveness, good drug loading capacity and a pH-responsive MTX release behavior, indicating their potential for targeted cancer therapy. These NPs displayed stable properties over at least 30 days, making them suitable for storage and transportation. Importantly, they showed excellent compatibility with blood components, suggesting their safe intravenous administration. In cytotoxicity tests, these nanocomposites demonstrated enhanced efficacy against breast cancer cells compared to free MTX. These findings collectively highlight their potential for clinical translation. Further \u003cem\u003ein vivo\u003c/em\u003e studies are warranted to validate their efficacy, biodistribution and safety profile, paving the way for their application in personalized and targeted cancer treatment strategies.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by FEDER, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i), Instituto de Salud Carlos III (FIS, Spain) (grant number PI19/01478), and\u0026nbsp;FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Spain (Grant No. P20_00346).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors' contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAna Medina-Moreno: data curation, formal analysis, investigation, methodology. Mazen M. El-Hammadi: formal analysis, validation, writing - original draft, writing - review \u0026amp; editing. Gema I. Martínez-Soler: data curation, formal analysis, investigation, methodology. Javier G. Ramos: data curation, formal analysis. Gracia García-García: data curation, formal analysis. José L. Arias: conceptualization, data curation, methodology, supervision, validation, writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData Availability Statement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKozminski P, Halik PK, Chesori R, Gniazdowska E. Overview of dual-acting drug methotrexate in different neurological diseases, autoimmune pathologies and cancers. Int J Mol Sci. 2020;21:3483. doi: 10.3390/ijms21103483.\u003c/li\u003e\n\u003cli\u003eCronstein BN, Aune TM. Methotrexate and its mechanisms of action in inflammatory arthritis. Nat Rev Rheumatol. 2020;16:145\u0026ndash;54. doi: 10.1038/s41584-020-0373-9.\u003c/li\u003e\n\u003cli\u003eAbolmaali SS, Tamaddon AM, Dinarvand R. A review of therapeutic challenges and achievements of methotrexate delivery systems for treatment of cancer and rheumatoid arthritis. Cancer Chemother Pharmacol. 2013;71:1115\u0026ndash;30. doi: 10.1007/s00280-012-2062-0.\u003c/li\u003e\n\u003cli\u003eKumar S, Garg NK, Jain A, Khopade A, Pandey P, Sawant KK. Nanocarriers mediated delivery of methotrexate is instrumental in treating auto-immune diseases and cancer. J Drug Deliv Sci Technol. 2023;88:104969. doi: 10.1016/j.jddst.2023.104969.\u003c/li\u003e\n\u003cli\u003eSolomon DH, Glynn RJ, Karlson EW, Lu F, Corrigan C, Colls J, Xu C, MacFadyen J, Barbhaiya M, Berliner N, Dellaripa PF, Everett BM, Pradhan AD, Hammond SP, Murray M, Rao DA, Ritter SY, Rutherford A, Sparks JA, Stratton J, Suh DH, Tedeschi SK, Vanni KMM, Paynter NP, Ridker PM. Adverse effects of low-dose methotrexate: a randomized trial. Ann Intern Med. 2020;172:369\u0026ndash;80. doi: 10.7326/M19-3369.\u003c/li\u003e\n\u003cli\u003eMukhtar M, Ezra Manicum AL, Shojaei Barjouei M, Eshaghi Malekshah R, Behzadmehr R, Rahdar A, Ghotekar S, Baino F. Nanocarriers for methotrexate delivery/codelivery in the frame of cancer diagnostics and treatment: a review. Front Biomater Sci. 2023;2:1200670. doi: 10.3389/fbiom.2023.1200670.\u003c/li\u003e\n\u003cli\u003eGiri BR, Yang HS, Song IS, Choi HG, Cho JH, Kim DW. Alternative methotrexate oral formulation: enhanced aqueous solubility, bioavailability, photostability, and permeability. Pharmaceutics. 2022;14:2073. doi: 10.3390/pharmaceutics14102073.\u003c/li\u003e\n\u003cli\u003eRay S, Joy M, Sa B, Ghosh S, Chakraborty J. pH dependent chemical stability and release of methotrexate from a novel nanoceramic carrier. RSC Adv. 2015;5:39482\u0026ndash;94. doi: 10.1039/C5RA03546E.\u003c/li\u003e\n\u003cli\u003eShariatifar H, Ranjbarian F, Hajiahmadi F, Farasat A. A comprehensive review on methotrexate containing nanoparticles; an appropriate tool for cancer treatment. Mol Biol Rep. 2022;49:11049\u0026ndash;60. doi: 10.1007/s11033-022-07782-7.\u003c/li\u003e\n\u003cli\u003eGavas S, Quazi S, Karpinski TM. Nanoparticles for cancer therapy: current progress and challenges. Nanoscale Res Lett. 2021;16:173. doi: 10.1186/s11671-021-03628-6.\u003c/li\u003e\n\u003cli\u003eNirmala MJ, Kizhuveetil U, Johnson A, Balaji G, Nagarajan R, Muthuvijayan V. Cancer nanomedicine: a review of nano-therapeutics and challenges ahead. RSC Adv. 2023;13:8606\u0026ndash;29. doi: 10.1039/d2ra07863e.\u003c/li\u003e\n\u003cli\u003eSun L, Liu H, Ye Y, Lei Y, Islam R, Tan S, Tong R, Miao YB, Cai L. Smart nanoparticles for cancer therapy. Signal Transduct Target Ther. 2023;8:418. doi: 10.1038/s41392-023-01642-x.\u003c/li\u003e\n\u003cli\u003eEl-Hammadi MM, Arias JL. Advanced engineering approaches in the development of PLGA-based nanomedicines. In: Aliofkhazraei M, editor. Handbook of Nanoparticles. Cham: Springer International Publishing AG; 2015. pp. 1009\u0026ndash;39.\u003c/li\u003e\n\u003cli\u003eRebollo R, Oyoun F, Corvis Y, El-Hammadi MM, Saubamea B, Andrieux K, Mignet N, Alhareth K. Microfluidic manufacturing of liposomes: development and optimization by design of experiment and machine learning. ACS Appl Mater Interfaces. 2022;14:39736\u0026ndash;45. doi: 10.1021/acsami.2c06627.\u003c/li\u003e\n\u003cli\u003eJim\u0026eacute;nez-L\u0026oacute;pez J, El-Hammadi MM, Ortiz R, Cayero-Otero MD, Cabeza L, Perazzoli G, Martin-Banderas L, Baeyens JM, Prados J, Melguizo C. A novel nanoformulation of PLGA with high non-ionic surfactant content improves in vitro and in vivo PTX activity against lung cancer. Pharmacol Res. 2019;141:451\u0026ndash;65. doi: 10.1016/j.phrs.2019.01.013.\u003c/li\u003e\n\u003cli\u003eEl-Hammadi MM, Delgado \u0026Aacute;V, Melguizo C, Prados JC, Arias JL. Folic acid-decorated and PEGylated PLGA nanoparticles for improving the antitumour activity of 5-fluorouracil. Int J Pharm. 2017;516:61\u0026ndash;70. doi: 10.1016/j.ijpharm.2016.11.012.\u003c/li\u003e\n\u003cli\u003eCabeza L, El-Hammadi MM, Ortiz R, Cayero-Otero MD, Jim\u0026eacute;nez-L\u0026oacute;pez J, Perazzoli G, Martin-Banderas L, Baeyens JM, Melguizo C, Prados J. Evaluation of poly (lactic-co-glycolic acid) nanoparticles to improve the therapeutic efficacy of paclitaxel in breast cancer. BioImpacts. 2022;12:515\u0026ndash;31. doi: 10.34172/bi.2022.23433.\u003c/li\u003e\n\u003cli\u003eEl-Hammadi MM, Small-Howard AL, Fern\u0026aacute;ndez-Ar\u0026eacute;valo M, Mart\u0026iacute;n-Banderas L. Development of enhanced drug delivery vehicles for three cannabis-based terpenes using poly(lactic-co-glycolic acid) based nanoparticles. Ind Crop Prod. 2021;164:113345. doi: 10.1016/j.indcrop.2021.113345.\u003c/li\u003e\n\u003cli\u003eEl-Hammadi MM, Small-Howard AL, Jansen C, Fernandez-Arevalo M, Turner H, Martin-Banderas L. Potential use for chronic pain: poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles enhance the effects of cannabis-based terpenes on calcium influx in TRPV1-expressing cells. Int J Pharm. 2022;616:121524. doi: 10.1016/j.ijpharm.2022.121524.\u003c/li\u003e\n\u003cli\u003eEl-Hammadi MM, Arias JL. Nanotechnology for vaginal drug delivery and targeting. In: Mozafari M, editor. Nanoengineered Biomaterials for Advanced Drug Delivery. Amsterdam: Elsevier Ltd.; 2020. pp. 647\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003eBhatia SN, Chen X, Dobrovolskaia MA, Lammers T. Cancer nanomedicine. Nat Rev Cancer. 2022;22:550\u0026ndash;6. doi: 10.1038/s41568-022-00496-9.\u003c/li\u003e\n\u003cli\u003eEl-Hammadi MM, Arias JL. Nanomedicine for vaginal drug delivery. In: Kesharwani P, Taurin S, Greish K, editors. Theory and Applications of Nonparenteral Nanomedicines. London: Academic Press: 2021. pp. 235\u0026ndash;57.\u003c/li\u003e\n\u003cli\u003eEl-Hammadi MM, Arias JL. Recent advances in the surface functionalization of PLGA-based nanomedicines. Nanomaterials (Basel). 2022;12:354. doi: 10.3390/nano12030354.\u003c/li\u003e\n\u003cli\u003eMadani F, Esnaashari SS, Bergonzi MC, Webster TJ, Younes HM, Khosravani M, Adabi M. Paclitaxel/methotrexate co-loaded PLGA nanoparticles in glioblastoma treatment: formulation development and in vitro antitumor activity evaluation. Life Sci. 2020;256:117943. doi: 10.1016/j.lfs.2020.117943.\u003c/li\u003e\n\u003cli\u003eJang JH, Jeong SH, Lee YB. Preparation and in vitro/in vivo characterization of polymeric nanoparticles containing methotrexate to improve lymphatic delivery. Int J Mol Sci. 2019;20:3312. doi: 10.3390/ijms20133312.\u003c/li\u003e\n\u003cli\u003eVakilinezhad MA, Amini A, Dara T, Alipour S. Methotrexate and curcumin co-encapsulated PLGA nanoparticles as a potential breast cancer therapeutic system: in vitro and in vivo evaluation. Colloids Surf B Biointerfaces. 2019;184:110515. doi: 10.1016/j.colsurfb.2019.110515.\u003c/li\u003e\n\u003cli\u003eNoreen S, Hasan S, Ghumman SA, Bukhari SNA, Ijaz B, Hameed H, Iqbal H, Aslam A, Elsherif MAM, Noureen S, Ejaz H. pH responsive Abelmoschus esculentus mucilage and administration of methotrexate: in-vitro antitumor and in-vivo toxicity evaluation. Int J Mol Sci. 2022;23:2725. doi: 10.3390/ijms23052725.\u003c/li\u003e\n\u003cli\u003eBarbosa AI, Costa Lima SA, Reis S. Development of methotrexate loaded fucoidan/chitosan nanoparticles with anti-inflammatory potential and enhanced skin permeation. Int J Biol Macromol. 2019;124:1115\u0026ndash;22. doi: 10.1016/j.ijbiomac.2018.12.014.\u003c/li\u003e\n\u003cli\u003eSaraei M, Sarvari R, Massoumi B, Agbolaghi S. Co-delivery of methotrexate and doxorubicin via nanocarriers of star-like poly(DMAEMA-block-HEMA-block-AAc) terpolymers. Polym Int. 2019;68:1795\u0026ndash;803. doi: 10.1002/pi.5890.\u003c/li\u003e\n\u003cli\u003eBashir SM, Ahmed Rather G, Patr\u0026iacute;cio A, Haq Z, Sheikh AA, Shah MZUH, Singh H, Khan AA, Imtiyaz S, Ahmad SB, Nabi S, Rakhshan R, Hassan S, Fonte P. Chitosan nanoparticles: a versatile platform for biomedical applications. Materials (Basel). 2022;15:6521. doi: 10.3390/ma15196521.\u003c/li\u003e\n\u003cli\u003eDesai N, Rana D, Salave S, Gupta R, Patel P, Karunakaran B, Sharma A, Giri J, Benival D, Kommineni N. Chitosan: a potential biopolymer in drug delivery and biomedical applications. Pharmaceutics. 2023;15:1313. doi: 10.3390/pharmaceutics15041313.\u003c/li\u003e\n\u003cli\u003eHarugade A, Sherje AP, Pethe A. Chitosan: a review on properties, biological activities and recent progress in biomedical applications. React Funct Polym. 2023;191:105634. doi: 10.1016/j.reactfunctpolym.2023.105634.\u003c/li\u003e\n\u003cli\u003ePuluhulawa LE, Joni IM, Elamin KM, Mohammed AFA, Muchtaridi M, Wathoni N. Chitosan-hyaluronic acid nanoparticles for active targeting in cancer therapy. Polymers (Basel). 2022;14:3410. doi: 10.3390/polym14163410.\u003c/li\u003e\n\u003cli\u003eGeethakumari D, Bhaskaran Sathyabhama A, Raji Sathyan K, Mohandas D, Somasekharan JV, Thavarool Puthiyedathu S. Folate functionalized chitosan nanoparticles as targeted delivery systems for improved anticancer efficiency of cytarabine in MCF-7 human breast cancer cell lines. Int J Biol Macromol. 2022;199:150\u0026ndash;61. doi: 10.1016/j.ijbiomac.2021.12.070.\u003c/li\u003e\n\u003cli\u003eSharifi-Rad J, Quispe C, Butnariu M, Rotariu LS, Sytar O, Sestito S, Rapposelli S, Akram M, Iqbal M, Krishna A, Kumar NVA, Braga SS, Cardoso SM, Jafernik K, Ekiert H, Cruz-Martins N, Szopa A, Villagran M, Mardones L, Martorell M, Docea AO, Calina D. Chitosan nanoparticles as a promising tool in nanomedicine with particular emphasis on oncological treatment. Cancer Cell Int. 2021;21:318. doi: 10.1186/s12935-021-02025-4.\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-\u0026Aacute;lvarez F, Garc\u0026iacute;a-Garc\u0026iacute;a G, Arias JL. A tri-stimuli responsive (maghemite/PLGA)/chitosan nanostructure with promising applications in lung cancer. Pharmaceutics. 2021;13:1232. doi: 10.3390/pharmaceutics13081232.\u003c/li\u003e\n\u003cli\u003eMohammed MA, Syeda JTM, Wasan KM, Wasan EK. An overview of chitosan nanoparticles and its application in non-parenteral drug delivery. Pharmaceutics. 2017;9:53. doi: 10.3390/pharmaceutics9040053.\u003c/li\u003e\n\u003cli\u003eAibani N, Rai R, Patel P, Cuddihy G, Wasan EK. Chitosan nanoparticles at the biological interface: implications for drug delivery. Pharmaceutics. 2021;13:1686. doi: 10.3390/pharmaceutics13101686.\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-\u0026Aacute;lvarez F, Garc\u0026iacute;a-Garc\u0026iacute;a G, Arias JL. A tri-stimuli responsive (maghemite/PLGA)/chitosan nanostructure with promising applications in lung cancer. Pharmaceutics. 2021;13:1232. doi: 10.3390/pharmaceutics13081232.\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-\u0026Aacute;lvarez F, Caro C, Garc\u0026iacute;a-Garc\u0026iacute;a G, Garc\u0026iacute;a-Mart\u0026iacute;n ML, Arias JL. Engineering of stealth (maghemite/PLGA)/chitosan (core/shell)/shell nanocomposites with potential applications for combined MRI and hyperthermia against cancer. J Mater Chem B. 2021;9:4963\u0026ndash;80. doi: 10.1039/D1TB00354B.\u003c/li\u003e\n\u003cli\u003eMedina-Moreno A, El-Hammadi MM, Arias JL. pH-dependent, extended release and enhanced in vitro efficiency against colon cancer of Tegafur formulated using chitosan-coated poly(\u0026epsilon;-caprolactone) nanoparticles. J Drug Deliv Sci Technol. 2023;86:104594. doi: 10.1016/j.jddst.2023.104594.\u003c/li\u003e\n\u003cli\u003eAmoozgar Z, Park J, Lin Q, Yeo Y. Low molecular-weight chitosan as a pH-sensitive stealth coating for tumor-specific drug delivery. Mol Pharm. 2012;9:1262. doi: 10.1021/mp2005615.\u003c/li\u003e\n\u003cli\u003eIshak RA, Awad GA, Zaki NM, El-Shamy Ael H, Mortada ND. A comparative study of chitosan shielding effect on nano-carriers hydrophilicity and biodistribution. Carbohydr Polym. 2013;94:669\u0026ndash;76. doi: 10.1016/j.carbpol.2013.01.072.\u003c/li\u003e\n\u003cli\u003eBaati T, Chaabani I, Salek A, Njim L, Selmi M, Al-Kattan A, Hosni K. Chitosan-coated ultrapure silicon nanoparticles produced by laser ablation: biomedical potential in nano-oncology as a tumor-targeting nanosystem. Nanoscale Adv. 2023;5:3044\u0026ndash;52. doi: 10.1039/d3na00253e.\u003c/li\u003e\n\u003cli\u003eElkomy MH, Ali AA, Eid HM. Chitosan on the surface of nanoparticles for enhanced drug delivery: a comprehensive review. J Control Release. 2022;351:923\u0026ndash;40. doi: 10.1016/j.jconrel.2022.10.005.\u003c/li\u003e\n\u003cli\u003eNur SG, Buket O, Sezgi K, Cagdas A, Cansu A, Guliz A, Habibe Y, Cigir BA, Yavuz D, Senay HS. Synthesis of methotrexate loaded chitosan nanoparticles and in vitro evaluation of the potential in treatment of prostate cancer. Anticancer Agents Med Chem. 2016;16:1038\u0026ndash;42. doi: 10.2174/1871520616666160101120040.\u003c/li\u003e\n\u003cli\u003eNogueira DR, Tavano L, Mitjans M, Perez L, Infante MR, Vinardell MP. In vitro antitumor activity of methotrexate via pH-sensitive chitosan nanoparticles. Biomaterials. 2013;34:2758\u0026ndash;72. doi: 10.1016/j.biomaterials.2013.01.005.\u003c/li\u003e\n\u003cli\u003eMazzotta E, De Benedittis S, Qualtieri A, Muzzalupo R. Actively targeted and redox responsive delivery of anticancer drug by chitosan nanoparticles. Pharmaceutics. 2019;12:26. doi: 10.3390/pharmaceutics12010026.\u003c/li\u003e\n\u003cli\u003eAl-Nemrawi N, Hameedat F, Al-Husein B, Nimrawi S. Photolytic controlled release formulation of methotrexate loaded in chitosan/TiO(2) nanoparticles for breast cancer. Pharmaceuticals (Basel). 2022;15:149. doi: 10.3390/ph15020149.\u003c/li\u003e\n\u003cli\u003eReddy LH, Arias JL, Nicolas J, Couvreur P. Magnetic nanoparticles: design and characterization, toxicity and biocompatibility, pharmaceutical and biomedical applications. Chem Rev. 2012;112:5818\u0026ndash;78. doi: 10.1021/cr300068p.\u003c/li\u003e\n\u003cli\u003eMater\u0026oacute;n Em, Miyazaki CM, Carr O, Joshi N, Picciani PHS, Dalmaschio CJ, Davis F, Shimizu FM. Magnetic nanoparticles in biomedical applications: a review. Appl Surf Sci Adv. 2021;6:100163. doi: 10.1016/j.apsadv.2021.100163.\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-\u0026Aacute;lvarez F, Garc\u0026iacute;a-Garc\u0026iacute;a G, Iglesias GR, Arias JL. A maghemite/PLGA (core/shell) nanostructure that may facilitate chemotherapy and antitumor hyperthermia. J Magn Magn Mater. 2024;589:171574. doi: 10.1016/j.jmmm.2023.171574.\u003c/li\u003e\n\u003cli\u003eEl-Hammadi MM, Arias JL. Iron oxide-based multifunctional nanoparticulate systems for biomedical applications: a patent review (2008 - present). Expert Opin Ther Pat. 2015;25:691\u0026ndash;709. doi: 10.1517/13543776.2015.1028358.\u003c/li\u003e\n\u003cli\u003eArias JL, Reddy LH, Couvreur P. Fe3O4/chitosan nanocomposite for magnetic drug targeting to cancer. J Mater Chem. 2012;22:7622\u0026ndash;32. doi: 10.1039/C2JM15339D.\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Garc\u0026iacute;a G, Caro C, Fern\u0026aacute;ndez-\u0026Aacute;lvarez F, Garc\u0026iacute;a-Mart\u0026iacute;n ML, Arias JL. Multi-stimuli-responsive chitosan-functionalized magnetite/poly(epsilon-caprolactone) nanoparticles as theranostic platforms for combined tumor magnetic resonance imaging and chemotherapy. Nanomedicine. 2023;52:102695. doi: 10.1016/j.nano.2023.102695.\u003c/li\u003e\n\u003cli\u003eKazemi S, Pourmadadi M, Yazdian F, Ghadami A. The synthesis and characterization of targeted delivery curcumin using chitosan-magnetite-reduced graphene oxide as nano-carrier. Int J Biol Macromol. 2021;186:554\u0026ndash;62. doi: 10.1016/j.ijbiomac.2021.06.184.\u003c/li\u003e\n\u003cli\u003eAl-Hussainawy MK, Sahb Mehdi Z, Jasim KK, Alshamsi HA, Saud HR, Kyhoiesh HAK. A single rapid route synthesis of magnetite/chitosan nanocomposite: competitive study. Results Chem. 2022;4:100567. doi: 10.1016/j.rechem.2022.100567.\u003c/li\u003e\n\u003cli\u003eJamir M, Borgohain C, Borah JP. Chitosan modified Fe3O4 nanoparticles for hyperthermia application. Mater Today Proc. 2022;65:2484\u0026ndash;9. doi: 10.1016/j.matpr.2022.04.176.\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Garc\u0026iacute;a G, L\u0026aacute;zaro-Callej\u0026oacute;n M, Fern\u0026aacute;ndez-\u0026Aacute;lvarez F, Iglesias GR, Arias JL. (Magnetite/poly(\u0026epsilon;-caprolactone))/chitosan (core/shell)/shell nanocomposites with potential applications in hyperthermia cancer therapy. J Magn Magn Mater. 2023;588:171500. doi: 10.1016/j.jmmm.2023.171500.\u003c/li\u003e\n\u003cli\u003eMoskvin M, Hunto\u0026scaron;ov\u0026aacute; V, Herynek V, Matou\u0026scaron; P, Michalcov\u0026aacute; A, Lobaz V, Zasońska B, \u0026Scaron;louf M, Seliga R, Hor\u0026aacute;k D. In vitro cellular activity of maghemite/cerium oxide magnetic nanoparticles with antioxidant properties. Colloids Surf B Biointerfaces. 2021;204:111824. doi: 10.1016/j.colsurfb.2021.111824.\u003c/li\u003e\n\u003cli\u003eFang K, Song L, Gu Z, Yang F, Zhang Y, Gu N. Magnetic field activated drug release system based on magnetic PLGA microspheres for chemo-thermal therapy. Colloids Surf B Biointerfaces. 2015;136:712\u0026ndash;20. doi: 10.1016/j.colsurfb.2015.10.014.\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Garc\u0026iacute;a G, Fern\u0026aacute;ndez-\u0026Aacute;lvarez F, Cabeza L, Delgado \u0026Aacute;V, Melguizo C, Prados JC, Arias JL. Gemcitabine-loaded magnetically responsive poly(epsilon-caprolactone) nanoparticles against breast cancer. Polymers (Basel). 2020;12:2790. doi: 10.3390/polym12122790.\u003c/li\u003e\n\u003cli\u003eMu\u0026ntilde;oz de Escalona M, S\u0026aacute;ez-Fern\u0026aacute;ndez E, Prados JC, Melguizo C, Arias JL. Magnetic solid lipid nanoparticles in hyperthermia against colon cancer. Int J Pharm. 2016;504:11\u0026ndash;9. doi: 10.1016/j.ijpharm.2016.03.005.\u003c/li\u003e\n\u003cli\u003eArias JL, L\u0026oacute;pez-Viota M, S\u0026aacute;ez-Fern\u0026aacute;ndez E, Ruiz MA, Delgado \u0026Aacute;V. Engineering of an antitumor (core/shell) magnetic nanoformulation based on the chemotherapy agent ftorafur. Colloids Surf A Physicochem Eng Aspects. 2011;384:157\u0026ndash;63. doi: 10.1016/j.colsurfa.2011.03.051.\u003c/li\u003e\n\u003cli\u003eSantos DP, Ruiz MA, Gallardo V, Zanoni MVB, Arias JL. Multifunctional antitumor magnetite/chitosan-l-glutamic acid (core/shell) nanocomposites. J Nanopart Res. 2011;13:4311\u0026ndash;23. doi: 10.1007/s11051-011-0378-z.\u003c/li\u003e\n\u003cli\u003eNasrazadani S, Hassani S. Modern analytical techniques in failure analysis of aerospace, chemical, and oil and gas industries. In: Makhlouf ASH, Aliofkhazraei M, editors. Handbook of Materials Failure Analysis with Case Studies from the Oil and Gas Industry. Amsterdam: Elsevier B.V. 2016. pp. 39\u0026ndash;54.\u003c/li\u003e\n\u003cli\u003eWang J, Sui M, Fan W. Nanoparticles for tumor targeted therapies and their pharmacokinetics. Curr Drug Metab. 2010;11:129\u0026ndash;41. doi: 10.2174/138920010791110827.\u003c/li\u003e\n\u003cli\u003eHe C, Hu Y, Yin L, Tang C, Yin C. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials. 2010;31:3657\u0026ndash;66. doi: 10.1016/j.biomaterials.2010.01.065.\u003c/li\u003e\n\u003cli\u003eVoon SH, Tiew SX, Kue CS, Lee HB, Kiew LV, Misran M, Kamkaew A, Burgess K, Chung LY. Chitosan-coated poly(lactic-co-glycolic acid)-diiodinated boron-dipyrromethene nanoparticles improve tumor selectivity and stealth properties in photodynamic cancer therapy. J Biomed Nanotechnol. 2016;12:1431\u0026ndash;52. doi: 10.1166/jbn.2016.2263.\u003c/li\u003e\n\u003cli\u003eMoraru C, Mincea M, Menghiu G, Ostafe V. Understanding the factors influencing chitosan-based nanoparticles-protein corona interaction and drug delivery applications. Molecules. 2020;25:4758. doi: 10.3390/molecules25204758.\u003c/li\u003e\n\u003cli\u003eWu P, He X, Wang K, Tan W, He C, Zheng M. A novel methotrexate delivery system based on chitosan-methotrexate covalently conjugated nanoparticles. J Biomed Nanotechnol. 2009;5:557\u0026ndash;64. doi: 10.1166/jbn.2009.1073.\u003c/li\u003e\n\u003cli\u003eBadran MM, Mady MM, Ghannam MM, Shakeel F. Preparation and characterization of polymeric nanoparticles surface modified with chitosan for target treatment of colorectal cancer. Int J Biol Macromol. 2017;95:643\u0026ndash;49. doi: 10.1016/j.ijbiomac.2016.11.098.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cancer targeted therapy, chitosan, core/shell nanoparticles, magnetic-driven nanoparticles, magnetite, Methotrexate, pH-responsive drug release.","lastPublishedDoi":"10.21203/rs.3.rs-4328624/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4328624/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMethotrexate successful therapy encounters various challenges in chemotherapy, such as poor oral bioavailability, low specificity, side effects and the development of drug resistances. In this study, we propose a dual-targeted nanocarrier comprising magnetite/chitosan nanoparticles for an efficient Methotrexate delivery. The synthesis of the particles was confirmed through morphological analysis using electron microscopy and elemental mappings via energy dispersive X-ray spectroscopy. These nanoparticles exhibited a size of ≈ 270 nm, a zeta potential of ≈ 24 mV, and magnetic responsiveness, as demonstrated by hysteresis cycle analysis and visual observations under a magnetic field. In addition, these core/shell particles displayed high stability, as evidenced by size and surface electric charge measurements, during storage at both 4 ºC and 25 ºC for at least 30 days. Electrophoretic properties were examined in relation to pH and ionic strength, confirming the stability. The nanoparticles demonstrated a pH-responsive drug release as observed by a sustained Methotrexate release over the next 90 h under pH ≈ 7.4, while complete release occurred within 3 h under acidic conditions (pH ≈ 5.5). In the \u003cem\u003eex vivo\u003c/em\u003e biocompatibility assessment, the magnetite/chitosan particles showed excellent hemocompatibility and no cytotoxic effects on normal MCF-10A and cancer MCF-7 cells. Furthermore, the Methotrexate-loaded nanoparticles significantly enhanced the antitumor activity reducing the half-maximal inhibitory concentration by ≈ 2.7-fold less compared to the free chemotherapeutic.\u003c/p\u003e","manuscriptTitle":"Magnetic and pH-Responsive Magnetite/Chitosan (Core/Shell) Nanoparticles for Dual-Targeted Methotrexate Delivery in Cancer Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-10 20:05:51","doi":"10.21203/rs.3.rs-4328624/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2024-06-01T10:28:27+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-05-02T00:03:22+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-01T18:15:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-30T05:56:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Drug Delivery and Translational Research","date":"2024-04-29T04:31:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"02fb2fd9-2245-429f-8fbd-abf2e194efbd","owner":[],"postedDate":"May 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-09T16:09:00+00:00","versionOfRecord":{"articleIdentity":"rs-4328624","link":"https://doi.org/10.1007/s13346-024-01701-y","journal":{"identity":"drug-delivery-and-translational-research","isVorOnly":false,"title":"Drug Delivery and Translational Research"},"publishedOn":"2024-09-05 16:04:54","publishedOnDateReadable":"September 5th, 2024"},"versionCreatedAt":"2024-05-10 20:05:51","video":"","vorDoi":"10.1007/s13346-024-01701-y","vorDoiUrl":"https://doi.org/10.1007/s13346-024-01701-y","workflowStages":[]},"version":"v1","identity":"rs-4328624","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4328624","identity":"rs-4328624","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}