Development of Dual-Responsive Magnetic/NIR-Triggered Multilayer Microcapsules for Controlled Methotrexate Delivery in Cancer Therapy

Development of Dual-Responsive Magnetic/NIR-Triggered Multilayer Microcapsules for Controlled 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 Development of Dual-Responsive Magnetic/NIR-Triggered Multilayer Microcapsules for Controlled Methotrexate Delivery in Cancer Therapy Samira Kariminia, Mojtaba Shamsipur This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7925880/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jan, 2026 Read the published version in Journal of Polymers and the Environment → Version 1 posted 13 You are reading this latest preprint version Abstract Near-infrared (NIR)-responsive drug delivery systems offer precise spatiotemporal control for targeted cancer therapy with minimal invasiveness. Here, we report the design of magnetic/NIR-responsive multilayer microcapsules via layer-by-layer assembly of biocompatible chitosan (CS) and carboxymethyl cellulose (CMC). Incorporation of magnetite (Fe₃O₄) and gold nanoparticles (Au NPs) into the microcapsule shells enabled both magnetic guidance and efficient NIR-triggered drug release. Methotrexate (MTX), a potent anti-cancer agent, was encapsulated with a high loading efficiency (68%). Systematic studies revealed the influence of bilayer number, MTX concentration, and environmental pH on drug loading and release kinetics. In vitro drug release was studied in different pH values and in the presence of an NIR irradiation. While the drug releases much faster in an acidic environment (pH 5.0) compared to physiological pH (7.4), it maintains stable release across both conditions, leading to uniform therapeutic delivery. Under NIR irradiation for15 min, up to 85% of the loaded MTX was released, while less than 7% was released when incubated in the dark for the same duration, significantly enhancing cytotoxicity against MCF-7 breast cancer cells. These dual-responsive microcapsules offer a versatile platform for targeted chemotherapy and photothermal therapy, highlighting their potential in minimally invasive cancer treatment. Drug delivery Multilayer microcapsule. Responsive drug release NIR irradiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Cancer treatment remains a major clinical challenge, as chemotherapy often limited by systemic toxicity, multidrug resistance, and non-specific drug delivery. To overcome these limitations, stimulus-responsive drug delivery systems have emerged as transformative approaches, offering precise, localized drug release triggered by internal or external stimuli such as pH, biomolecules, magnetic fields, or light[ 1 ]. Among these, near-infrared (NIR) light-responsive systems are particularly attractive due to their non-invasive application, high tissue penetration depth, and minimal damage to healthy cells [ 2 ]. Multilayer microcapsules, fabricated via the layer-by-layer (LbL) technique, have demonstrated unique advantages in stimuli-responsive drug delivery owing to their tunable hollow structures, material versatility, and ability to incorporate a variety of functional nanoparticles[ 3 ] [ 4 ]. The use of natural polysaccharides and their derivatives in capsule formulation is a promising avenue for drug delivery applications Chitosan (CS) (Fig. 1 a), (1–4)2-amino 2-deoxy b-D glucan, a polysaccharide abundantly present in crustacean shells, is derived from partially deacetylated chitin. Chitosan (CS) is a widely recognized biopolymer with excellent biocompatibility and biodegradability, making it ideal for use in drug delivery systems [ 5 ] , [ 6 ]. The polyanionic polysaccharide carboxymethyl cellulose (CMC) (Fig. 1 b) possesses a valuable combination of properties: biocompatibility, biodegradability, and low cost. These characteristics, along with its bioavailability, make CMC a promising candidate for the development of sustainable drug delivery systems[ 7 ].Additionally, integrating magnetite (Fe₃O₄) and gold (Au) nanoparticles into the capsule shells allows for dual-functional systems,achieving both magnetic targeting and efficient photothermal response under NIR irradiation[ 8 ]. Au nanoparticles have caused the localized heating to lead to the destruction of microcapsules. This innovative approach has proven effective in releasing encapsulated peptides, proteins, and model drugs, making it a promising technique for precise and targeted drug delivery[ 9 ]. This capability opens up possibilities for remotely controlled drug release and targeted delivery systems[ 10 , 11 ].Methotrexate (MTX) (Fig. 1 c), a widely used chemotherapeutic agent, effectively inhibits DNA synthesis but is associated with significant off-target toxicity including leukopenia, hepatotoxicity, nephrotoxicity, and bone marrow suppression[ 12 ]. These side effects significantly limit the effectiveness of the treatment[ 13 , 14 ] . In this study, we propose that magnetic and NIR-responsive multilayer microcapsules constructed from biocompatible CS and CMC polymers can provide an effective platform for MTX delivery. These microcapsules were synthesized via layer-by-layer electrostatic assembly of CS and CMC on a sacrificial CaCO 3 template, incorporating Fe 3 O 4 and Au nanoparticles for magnetic targeting and near-infrared (NIR) light-triggered release, respectively. Following EDTA-mediated template removal, the resulting hollow microcapsules were loaded with MTX at various concentrations and pH values. The in vitro cytotoxicity of the MTX-loaded multilayer microcapsules against MCF-7 breast cancer cells was evaluated upon magnetic targeting and NIR irradiation (780 nm). The findings highlight the potential of these microcapsules as advanced platforms for minimally invasive cancer therapy. 2. Experimental 2.1. Materials Carboxymethyl cellulose (CMC, M w ~70 kDa, degree of substitution = 0.9), Chitosan (CS, M w ~10kDa, deacetylation degree: 91%)), Hydrogen tetrachloroaurate trihydrate (HAuCl 4 .3H 2 O), Ferric chloride hexahydrate (FeCl 3 .6H 2 O), Ferrous chloride tetrahydrate (FeCl 2 .4H 2 O) and MTT assay (3-[4,5- dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide) were purchased from Sigma Aldrich. Citric acid, Ethylenediaminetetraacetic acid disodium salt (EDTA), Sodium chloride (NaCl), Ammonium hydroxide (NH 4 OH), Calcium chloride (CaCl 2 ), Sodium hydroxide (NaOH), Sodium carbonate (Na 2 CO 3 ), were bought from Merck. The MCF-7 human breast cancer cell line (RRID: CVCL_0031) was obtained from the Iranian Biological Resource Center (IBRC), Tehran, Iran. The cells were tested for mycoplasma contamination using a PCR-based Mycoplasma Detection Kit (Pishtaz Teb Diagnostics, Iran) and were confirmed to be mycoplasma-free. 2.2. Synthesis of citrate coated Fe 3 O 4 nanoparticles As described previously, the co-precipitation method was used for preparation of the Fe 3 O 4 nanoparticle (NP) using Fe 2+ and Fe 3+ ions[ 15 ]. In a typical reaction, 25 mL FeCl 3 .6H 2 O (0.2 M) and FeCl 2 .4H 2 O (0.1 M) solutions were prepared and mixed at water bath (60°C). The co-precipitation of Fe 3 O 4 NPs was carried out in a three-neck round-bottom flask under nitrogen atmosphere. To initiate the formation process of Fe 3 O 4 NPs, a solution of ammonium (5 mL of 25% NH 3 ) was introduced. Following that, a solution of 0.1g citric acid in 20 mL of water was added, and the temperature was raised to 95ºC. The Fe 3 O 4 NPs were then exposed to citric acid treatment for 1 hour, resulting in the development of negative charges on their surfaces. The black precipitate was carefully separated through sedimentation using an external magnet, followed by multiple rinses with water to ensure thorough purification. 2.3. Preparation of Au nanoparticles Au NPs were prepared by the protocol outlined by Kimura et al[ 16 ]. To summarize, in 20 mL of deionized water, a solution was prepared by dissolving 0.5 mL of HAuCl 4 (20.8 mmol/l). The resulting faint yellowish solution was then heated to a boiling point. During this process, 1 mL of sodium citrate (0.5%) was added to solution and was vigorously stirred. After 30-minute stirring, yellowish color of the solution was changed to red color, indicating the successful synthesize of Au NPs. 2.4. Preparation of CaCO 3 microparticles doped with CMC CaCO 3 (CMC), also known as calcium carbonate microparticles doped with CMC, were produced following the previously described method [ 17 ]. A solution of Ca(NO 3 ) 2 (100 mL, 0.025 M) was combined with CMC solution (2 mL, 5% w/v). The process began by combining the aforementioned solution with of Na 2 CO 3 (100 mL, 0.025 M) solution at ambient temperature. The CaCO 3 microparticles were obtained by vigorously agitating the precipitate using a magnetic stirrer for a duration of 30 seconds. After sedimentation, they were washed three times with distilled water and subsequently underwent air drying. This resulted in the formation of the CaCO 3 (CMC) hybrid microparticles. 2.5. Fabrication of multilayer CS/CMC microcapsules functionalized with Au and Fe 3 O 4 NPs .The synthesize of the microcapsules involved a layer-by-layer template-assisted assembly[ 18 ]. The cores were subsequently removed to create the hollow magnetic multilayer CS/CMC microcapsules[ 19 ]. Briefly, the microparticles of CaCO 3 (CMC) were dispersed in a solution of CS (2 mg/mL) containing 0.5 M NaCl. The mixture was then gently shaken for a duration of 15 minutes to establish the CS layer on template. Next, the coated microparticles underwent separation through centrifugation (10,000 rpm, 1 min). Subsequently, they were washed three times using a 0.5 M NaCl solution at a pH of 5.0. After being washed with distilled water, the precipitate was dispersed in solution of CMC (1mg/mL) and was shaken for 15 minutes. The microparticles were separated using centrifugation and were subsequently rinsed with a 0.5 M NaCl solution at pH 5.0 for three times. Using a similar method, the negatively charged CMC layer was replaced with Fe 3 O 4 NPs and Au NPs separately. Then unabsorbed NPs were eliminated through magnetic decantation and washing steps. Consequently, different numbers of bilayers (ranging from 8 to 12) were generated by repeating bilayer coating, while chitosan was selected as the outer shell. The 1% glutaraldehyde (GA) solution was used to cross-link the magnetic multilayer CS/CMC microcapsules at room temperature for a duration of 12 hours [ 20 ]. Hollow microcapsules were prepared by adding EDTA solution (0.2 M at pH 7.0 for 15 min incubation) to removal of template. The hollow microcapsules were subjected to three cycles of rinsing using EDTA, followed by three cycles of rinsing with water. Then the samples were subjected to a drying process in an oven at a temperature of 50°C for 1 hour. Subsequently, they were stored for future investigations. The final shell composition of the microcapsule was (CS/CMC) 4 (CS/Fe 3 O 4 ) 2 (CS/Au NP) 2 (CS/CMC) 2 CS. The steps of multilayer microcapsule preparation and drug loading are drawn schematically (Schem1) 2.6. MTX loading into magnetic multilayer CS/CMC microcapsules The preparation of the magnetic multilayer CS/CMC microcapsules discussed in the preceding section was carried out with the exception that, prior to the addition of GA, we introduced magnetic CS/CMC microcapsules (1 mg) into 5 mL MTX solution with different concentrations (10–200 ppm) and different pH values. The mixture was magnetically stirred at ambient temperature for a duration of 24 hours. MTX was incorporated into the magnetic CS/CMC microcapsule by physical adsorption. A 0.50 T magnet was used to separate the unloaded MTX from MTX loaded multilayer CS/CMC microcapsules. The concentration of MTX in the supernatant was measured using a UV-Vis spectrophotometer with absorbance at 303 nm. Calibration curve was determined for pure MTX (A = 0.0215C ppm -0.0278) with linear concentration range (5–50 ppm). The loading efficiency (LE) of MTX was determined using the below equation: LE (%) = \(\:\frac{\mathbf{t}\mathbf{o}\mathbf{t}\mathbf{a}\mathbf{l}\:\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{c}.\:\:\mathbf{o}\mathbf{f}\:\mathbf{d}\mathbf{r}\mathbf{u}\mathbf{g}\:-\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{c}.\:\:\mathbf{o}\mathbf{f}\:\mathbf{u}\mathbf{n}\mathbf{b}\mathbf{o}\mathbf{u}\mathbf{n}\mathbf{d}\mathbf{e}\mathbf{d}\:\mathbf{d}\mathbf{r}\mathbf{u}\mathbf{g}}{\mathbf{t}\mathbf{o}\mathbf{t}\mathbf{a}\mathbf{l}\:\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{c}.\:\:\mathbf{o}\mathbf{f}\:\mathbf{d}\mathbf{r}\mathbf{u}\mathbf{g}}\:\times\:100\) (Eq. 1) 2.7. Drug release study The magnetic multilayer CS/CMC microcapsules containing MTX were evenly distributed in a 5 mL buffer solution (0.1M, pH 5.0 and 7.4) and subjected to incubation at 37 ◦ C with gentle agitation. This process was carried out both with and without NIR irradiation, using an output power of 200 mW, at specific time intervals for drug release. The UV-Vis spectrophotometer was utilized to measure the quantity of drug released at 303 nm. The MTX concentration was measured with the help of the standard calibration curve and the cumulative drug release (%) was calculated using the following equation: Cumulative drug release (%) = \(\:\frac{\varvec{m}\varvec{g}\:\left(\varvec{d}\varvec{r}\varvec{u}\varvec{g}\:\varvec{r}\varvec{e}\varvec{l}\varvec{e}\varvec{a}\varvec{s}\varvec{e}\varvec{d}\right)}{\varvec{m}\varvec{g}\:\left(\varvec{t}\varvec{o}\varvec{t}\varvec{a}\varvec{l}\:\varvec{d}\varvec{r}\varvec{u}\varvec{g}\right)}\:\times\:100\:\:\) (Eq. 2) 2.8. Cytotoxicity investigation For cytotoxicity investigation, human breast cancer MCF-7 cell line was used. The cell line was initially cultured in 96 well microplate and were kept in a humid atmosphere (95% air and 5% CO 2 ) at 37°C and the medium was refreshed. After this time, the cells were incubated in medium supplemented with different concentrations of MTX and To regulate the crystallization and growth patterns of the CaCO3 microparticles throughout the preparation procedure, researchers introduced carboxymethyl cellulose (CMC) into the reaction mixture. CMC is a polysaccharide with a negative charge, which was incorporated to achieve the formation of spherical microparticles of CaCO3. This addition of CMC helped in controlling the shape and size of the synthesized microparticles. Additionally, the presence of CMC in the reaction mixture has the added benefit of preserving the biocompatibility of the synthesized microparticles. synthesized hollow microcapsules (10–40 ppm) for 48 h. During the photothermal treatments, a NIR light with an intensity of 200mW was used to irradiate the cells for 15min. After a 48-hour incubation period, MTT (5mg/mL in phosphate buffered saline (PBS)) was introduced to each well and allowed to incubate for 4 h at 37 ◦ C. To disperse the formazan crystals, the supernatant containing MTT was extracted and subsequently, 100 µl of dimethyl sulfoxide (DMSO) was introduced. The plate was subsequently stored at ambient temperature for a duration of 30 minutes. The absorbance of the solution was determined at \(\:\varvec{\lambda\:}=570\:\mathbf{n}\mathbf{m}\) using a plate reader (Awareness), with background subtraction performed at \(\:\:\varvec{\lambda\:}=630\:\mathbf{n}\mathbf{m}\) . 2.9. Characterization The Philips instrument, Model XL-30, was utilized to conduct SEM analysis (accelerating voltage of 26 kV). The TEM images were captured by employing an FEI TECNAI transmission electron microscope (accelerating voltage of 120 kV). Particle size distributions of Fe 3 O 4 and Au NPs and the zeta-potential of the CaCO 3 (CMC) microparticles after each layer coating was determined by a Zeta-sizer (Malvern Instruments Ltd.). The CaCO 3 (CMC) microparticles were analyzed using the Bernauer-Emmett-Teller technique to determine their specific surface area and pore size distribution. The surface analyzer model (BEL Japan) was utilized to conduct this measurement via N 2 adsorption desorption at -196 ◦ C. X-ray diffractometer (Philips) equipped with Cu Kα radiation were used to record the XRD patterns. FT-IR spectrometer (Bruker, ALPHA, Germany) in the range of 4000–400 cm − 1 was used to characterize the structure of synthesized materials. UV-Vis spectrophotometer (Agilent 8453) was utilized to record the absorption spectra in the UV-visible range. A laser beam operating at a wavelength of 780 nm in the near infrared (NIR) range, with a power output of 200 mW was utilized. 3. Results and discussion 3.1. Synthesize and characterization of magnetic multilayer CS/CMC microcapsule The SEM micrographs (Fig. 2 a and b) reveal that the CaCO 3 (CMC) microparticles possess a regular spherical structure with approximately 500 nm in size. Furthermore, the SEM images (Fig. 2 c) provide evidence of their porous morphology. In Fig. 2 d, the XRD pattern reveals that the microparticles of CaCO 3 (CMC) primarily consisted of vaterite, with a small presence of calcite [ 21 ]. The presence of carboxymethyl cellulose (CMC) in preparation of CaCO3 microparticles hindered the recrystallization process[ 22 ]. Hence, the inclusion of carboxymethyl cellulose (CMC) in the CaCO 3 microparticles significantly enhanced their stability compared to the particles without any additives[ 23 ]. The pore size distribution of CaCO 3 (CMC) was analyzed using the BJH method, as shown in Fig. 3 a. The results revealed that the average pore diameter of the microparticles was 5.6 nm. Figure 3 b presents additional evidence from nitrogen adsorption-desorption measurements, showing a significant increase in surface area, rising from 8.8 m²/g for CaCO 3 microparticles without CMC to 128.4 m²/g.[ 24 ]. This indicates that the presence of CMC greatly enhances the surface area and porosity of the CaCO 3 microparticles. The adsorption of CMC polymer molecules onto the carbonate surface causes a loosening of the microparticles' packing, resulting in an increase in surface area. To enhance the properties of CaCO 3 microparticles, a coating consisting of 10 bilayers was applied. The coating comprised of alternating layers of chitosan (CS) with a positive charge and sodium carboxymethyl cellulose (CMC), Fe 3 O 4 , and Au NPs with negative charge. This multi-layer coating aimed to improve the functionality and performance of the microparticles to achieve high charges for both CS and CMC, the polymer solutions were adjusted to an optimal pH of 5.0 using 1.0 M NaOH or HCl. This pH value was chosen as it ensures that both CS and CMC exhibit a high level of charge[ 25 ]. Au NPs were incorporated into microcapsules for the purpose of thermal therapy application. The characterization of the Au NPs using TEM and DLS techniques revealed that they possess a diameter ranging from 6 to 10 nm. (Fig. 4 a,b). The Au NPs displayed the peak absorption at 560 nm in the UV-Vis spectrum, as observed in Fig. 4 c. According to zeta potential measurements, the carboxyl groups of sodium citrate result in a negative surface charge of the Au NPs, exhibiting a zeta potential of -23.5 mV. Also, in order to achieve remote control and precise manipulation of microcapsules towards the desired location, Fe 3 O 4 NPs were synthesized and incorporated as a middle layer of multilayer microcapsules. As shown in the scanning electron microscopy (SEM) image (Fig. 4 d), the Fe 3 O 4 NPs exhibited a spherical morphology, possessing an average diameter ranging from 50 to 80 nm. The size of NPs was further verified with DLS analysis (Fig. 4 e). To verify that the Fe 3 O 4 was successfully prepared and coated with citrate, FTIR spectra of the Fe 3 O 4 and citrate coated Fe 3 O 4 were measured (Fig. 4 f). In citric acid, the peak at 1715 cm -1 , which corresponds to the C = O vibration, shifts to 1618 cm -1 when citric acid is coated onto the magnetite surface. The Fe 3 O 4 NPs exhibited a zeta potential of -18.3 mV upon measurement, indicating that the negative surface charge of Fe 3 O 4 NPs is generated by carboxyl groups of sodium citrate. With introducing Fe 3 O 4 NPs into microcapsules, their appearance was changed (Fig. 5 a (1) and (2)) and their density increased; resulting in more rapid sedimentation from a he utilization of a magnet in the process can further expedite the sedimentation of microcapsules, resulting in a faster settling (Fig. 5 a (3) and (4)). As shown in Fig. 5 b, when the zeta potential was measured at pH 5.0 for different coating steps, a reversal of the surface charge from positive to negative was observed. The adsorption of polyelectrolytes onto the surface is confirmed by this reversal of zeta potential. Since CMC is negatively charged, the obtained microparticles exhibited a zeta potential of -23.0 mV. The presence of alternating positive and negative charge potential variations on the surface of the capsules indicates that the polycation CS and polyanion CMC have been successfully coated on the CaCO 3 (CMC) microparticles in an alternating manner[ 26 ]. Finally, the CS outer layer of multilayer microcapsules was cross-linked by glutaraldehyde. Previous studies have shown that glutaraldehyde undergoes a reaction with materials that contain primary amine groups, such as chitosan, certain peptides, and polypeptides [ 27 ]. The result of this reaction is the formation of covalently cross-linked polymers. After the required number of layers were loaded, the template was removed using ethylenediaminetetraacetic acid (EDTA), resulting the hollow multilayer microcapsules. As shown in SEM image (Fig. 2 e), the prepared multilayer microcapsules showed a spherical shape and smooth surface morphology. The addition of different layers of polymers onto the porous CaCO 3 (CMC) microparticles transforms the rough surface into a smoother one. This is attributed to the slight agglomeration that occurs between the oppositely charged polyelectrolytes, which act as bridges between each other, promoting adhesion. The SEM image of the hollow multilayer CS/CMC microcapsule (Fig. 2 f) showed CaCO 3 template has been completely removed. BET analysis of the hollow multilayer microcapsules (Fig. 3 c) reveals a mean pore diameter of 3.6 nm, with a porosity of approximately 42%. To analyze the magnetic multilayer CS/CMC microcapsules, we employed FT-IR spectroscopy for characterization purposes. IR spectra of CaCO 3 microparticles, CS, CMC, magnetic multilayer CS/CMC microcapsules are shown in Fig. 6 . The spectral analysis of the CaCO 3 microparticles shows significant absorption peaks at 1380 cm -1 , 1010 cm -1 , 877 cm -1 , and 746 cm -1 . These peaks are characteristic modes of vibration for carbonate[ 28 ].With respect to CS spectrum, the N-H and O-H stretching vibration is responsible for the distinctive absorption band observed at 3450 cm -1 and the peaks at 1649 and 1603 cm -1 are attributed to the amide I and II bands[ 29 ]. In the spectrum of CMC), the O–H stretching vibration of the OH group is responsible for a wide band observed at 3419cm -1 , while the C–H stretching vibration of the CH 2 and CH 3 groups is indicated by the band at 2920 cm -1 .The COO group can be associated with the asymmetric and stretching vibrations, which are indicated by the absorption peaks observed at 1616 cm -1 and 1423 cm -1 , respectively[ 30 ]. For magnetic multilayer CMC/CS microcapsules, compared to the spectra of CS and CMC, The characteristic bands of -NH 3 + and -COO - , which represent the absorption of N-H bonds of primary amine group and COO group respectively, were observed at 1412 and 1597 cm -1 . These findings indicate the presence of a strong electrostatic interaction between the carboxylic acid salts (-COO - ) and the amino groups (-NH 3 + ) within the microcapsule[ 31 ]. The distinguishing characteristic of the magnetic multilayer CMC/CS microcapsules' spectrum is the prominent peak observed at 593 cm -1 , which correspond to the vibration of the Fe-O bond. 3.2. MTX loading studies By simple incubation of the hollow microcapsule in MTX solution, the MTX was loaded into the magnetic multilayer CS/CMC microcapsule. The observed phenomenon of a smoother surface on the coated porous CaCO 3 (CMC) microparticles can be attributed to the strong interaction between the carboxyl and amino groups of drugs with the CS and CMC polymers. This interaction leads to the formation of bridges between the oppositely charged layers, resulting in the agglomeration of the different layers and a smoother surface. To investigate the interaction between MTX and magnetic multilayer CS/CMC microcapsules, the FTIR spectra of magnetic multilayer CS/CMC microcapsules were examined both before and after MTX loading. As shown in Fig. 6 , for MTX, the main peaks of COOH at 1693cm -1 , -CONH at 1643cm -1 , aryl system at 1543cm -1 and 1500cm -1 and aromatic ring system at 830cm -1 are present. The N-H group is indicated by the peak at 3380 cm -1 . The stretching vibrations of the C-C or C-H bond are represented by peaks at 1490 and 1209 cm -1 [ 32 , 33 ]. The spectral group frequencies of MTX-loaded magnetic multilayer CS/CMC microcapsules (Fig. 6 ) were characterized at 2507cm -1 and 2928cm -1 for C-H vibrations. The MTX-loaded microcapsules exhibit a higher wavenumber (3430 cm − 1 ) for the N-H stretching peak, as different with free MTX (3380 cm − 1 ). This observation indicates an interaction between the NH 2 group of free MTX and the COOH group of the microcapsule, leading to a shift in the peak. The vibration absorptions at 1643 cm − 1 attributed to amide of free MTX shifted to a higher wavenumber (1673 cm − 1 ) as shown in Fig. 6 , further confirms the interaction of COOH and NH 2 groups. Figure 7 a compares the UV-Vis absorption intensities of the DOX peak at 303 nm before and after the drug-loading process. Various factors influencing the drug loading capacity are examined in the following sections. 3.2.1. The impact of layer thickness on loading efficiency of MTX In this work, we investigated the effect of layer thickness on the loading efficiency of MTX. The thickness of the layers was identified as a crucial factor in determining the loading efficiency of the drug. By examining the relationship between layer thickness and drug loading, we aimed to gain insights into optimizing the loading process and enhancing the effectiveness of MTX delivery. Figure 7 b exhibits that the number of coated layers have impact on the MTX loading efficiency (The MTX concentration remained constant at 50 ppm). The loading efficiency of MTX was maximum and 42% when 10 bilayers were applied onto the multilayer microcapsules' surface. The study found that as the number of layers increases (up to 10), the drug loading efficiency does not change and remains constant. This indicates that 10 bilayers are the ideal choice for synthesizing microcapsules with high loading efficiency. 3.2.2. The impact of different concentrations of MTX on loading efficiency To investigate the impact of different concentrations of MTX on drug loading efficiency, the magnetic CS/CMC microcapsules were combined with MTX solution of varying concentrations. As shown in Fig. 7 c, the maximum loading efficiency of MTX into microcapsules is determined 68% at concentration of 90 ppm. This finding suggests that the drug loading efficiency is greatly influenced by the concentration of MTX. At higher feeding concentration of MTX, the loading efficiency was decreased because of insufficient functional groups in the polymer concentration. 3.2.3. The impact of different pH values on loading efficiency The MTX loading efficiencies of magnetic CS/CMC multilayer microcapsule solutions were evaluated across a pH range of 2.0 to 6.0 (Fig. 7 d). The synthesized magnetic multilayer CS/CMC microcapsule exhibited a greater drug loading efficiency at pH 5.0 thus we select this pH for further experiments. 3.3. NIR- and pH-simulated MTX release from the magnetic multilayer CS/CMC microcapsules The investigation of the release of MTX from the magnetic multilayer CS/CMC microcapsules was conducted under physiological conditions (37ºC) with and without NIR irradiation. The experiments for release were carried out at pH levels of 5.0 and 7.4, considering the fact that tumor tissue has acidic pH levels, whereas normal tissue maintains a pH of around 7.4. This allowed the researchers to assess how the microcapsules respond to different pH conditions and evaluate the potential for targeted drug delivery in tumor tissues. 3.3.1. In vitro drug release under different pH values Drug release rate from magnetic multilayer CS/CMC microcapsule was slow and sustained to more than 35 h (Fig. 8 a). The drug release profiles of carrier were compared at pH 5.0 and pH 7.4. The results are obviously indicated that drug release at an acidic pH of 5.0 was much faster than physiological pH, emphasizing that drug release from microcapsule is controlled by pH, meaning the smaller toxicity of the MTX loaded magnetic multilayer microcapsule to normal tissues (pH 7.4). This represents advantages of a safe system for drug delivery. 3.3.2. Drug release investigation under NIR irradiation Au NPs have been employed as agents for photothermal conversion in the field of photothermal therapy due to their high near-infrared (NIR) light absorption coefficients [ 34 ]. Figure 8 b indicate the photothermal effects of water, Au NPs and magnetic multilayer CS/CMC microcapsule under NIR irradiation (200mW cm − 2 ). The photothermal effects of these materials can be summarized as follows: water does not exhibit significant photothermal effects under NIR irradiation, as it has a low absorption coefficient for NIR light. Au NPs exhibit efficient photothermal heating capabilities when exposed to NIR light. Due to presence of Au NPs, the magnetic multilayer CS/CMC microcapsule display photothermal properties that can be influenced by the presence of NIR light. The Au NPs and multilayer CS/CMC microcapsules show promising photothermal properties, making them suitable for applications in cancer treatment and other medical fields. These findings indicated that the produced heat caused by Au NPs is sufficient to fulfill the requirement for enhancing near-infrared (NIR) responsive drug release[ 35 ]. When irradiation time is increased, the drug release increases (Fig. 8 c). After approximately 15 min of irradiation, up to 85% of the loaded MTX was released, while less than 7% was released when incubated in the dark for the same duration. Additionally, the temperature increase followed a consistent pattern, with a rapid initial rise and then reaching a plateau after approximately 40 minutes of exposure to irradiation. The release of MTX from the microcapsules is triggered by the temperature increase and collapse of the microcapsules resulting from the photothermal effect of Au NPs upon NIR irradiation. The study findings suggest that a duration of 15 minutes of irradiation is sufficient for the complete rupture of the capsules and the subsequent release of the drug. Also, in our work, a cross-linking agent called GA was introduced to increase the stability of the multilayer coating on the template using the LbL technique. However, GA is not able to withstand high temperatures. When the Au NP within the microcapsule shells were exposed to NIR light, the temperature inside the shells increased significantly. As a result, the aldehyde groups found in GA was degraded, leading to the instability and degradation of the multilayers of microcapsule[ 36 ]. Consequently, the loaded MTX was liberated from the microcapsules. The responsiveness of our system to external near-infrared (NIR) stimulus surpasses that of simple diffusion-based release systems. This finding highlights the significant potential of our system as a drug delivery system that is both highly responsive and easily controllable. 3.4. MTT assay Figure 9 a shows the cell viability of the bare and MTX-loaded magnetic multilayer CS/CMC microcapsules in the concentration range of 10 to 40 ppm without/with the irradiation of 200 mw NIR for 15 min. Clearly, without NIR irradiation, the drug-free magnetic multilayer showed low cytotoxicity against the MCF-7 cancer cells, in while with 15 min of NIR irradiation, the toxicity of drug-free magnetic multilayer CS/CMC microcapsules significantly enhanced and 32–65% of tumor cells were killed in the concentration range of 10 to 40 ppm. However, the cytotoxicity remained largely unaffected in the control experiment (with and without NIR irradiation) (Fig. 9 b), suggesting that the presence of magnetic multilayer CS/CMC microcapsules is essential for the observation of the photothermal effect. This result emphasizes that the prepared magnetic multilayer CS/CMC microcapsules have the potential to serve as a highly effective photothermal agent for eradicating tumor cells when exposed to NIR irradiation. Under irradiation of NIR, the prepared magnetic multilayer CS/CMC microcapsules can serve as a highly effective photothermal agent, capable of effectively eliminating tumor cells. Their unique properties make them ideal for targeted photothermal therapy in various biomedical applications. For MTX-loaded magnetic multilayer CS/CMC microcapsules, without the assistance of NIR irradiation, the cell viability only reduced by 88–65% as the concentration increased from 5 to 40 ppm, which is understandable because of a little release of the loaded MTX the first 24 h. In contrast, with NIR irradiation for 15 min, for the MTX-loaded magnetic multilayer microcapsules in the same concentration range, cell viabilities decreased from 50 to 16%. A significant increase in the cytotoxicity of MTX-loaded magnetic multilayer CS/CMC microcapsules against cancer cells can be seen under 15 min of NIR radiation that indicates the developed magnetic multilayer CS/CMC microcapsules could be used in both chemo- and photothermal treatment, leading to a highly effective therapeutic outcome. This combined approach offers great potential for achieving excellent therapeutic efficacy in various biomedical applications. 4. Conclusion This study successfully fabricated novel biocompatible hollow magnetic multilayer CS/CMC microcapsules incorporating Fe 3 O 4 and Au NPs by LbL technique. These microcapsules exhibited precise magnetic guidance and demonstrated high sensitivity to NIR irradiation, leading to a significant enhancement in drug release compared to diffusion-based systems. From the cellular cytotoxicity study, under NIR irradiation, MTX-loaded magnetic multilayer CS/CMC microcapsules showed higher toxicity than cytotoxicity of drug-free magnetic multilayer CS/CMC microcapsules. This platform, combining magnetic targeting, NIR-triggered drug release, and photothermal therapy capabilities, holds significant promise for advancing targeted cancer treatment and other biomedical applications. 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16:15:08","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":70934,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/7292ee36fafc4d3a9816e783.png"},{"id":95566142,"identity":"ae53def3-c40a-4d1f-9177-0b85efab6ad7","added_by":"auto","created_at":"2025-11-10 16:18:08","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33740,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/0b06048427032c47e5d11aa1.png"},{"id":95566140,"identity":"fd3bd2d3-49fe-4640-94fb-1463f051f73c","added_by":"auto","created_at":"2025-11-10 16:18:08","extension":"xml","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103202,"visible":true,"origin":"","legend":"","description":"","filename":"f67237d33d6943539551169eee67c0ca1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/9d635ca94cfacd1bb846d284.xml"},{"id":95566143,"identity":"c5baf9e2-6946-4d1b-96f8-8e70ed111f9f","added_by":"auto","created_at":"2025-11-10 16:18:08","extension":"html","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109097,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/bf63884faa709d7299ddba23.html"},{"id":95566102,"identity":"2ceadbd9-8d87-43a4-a05f-0660305d24c6","added_by":"auto","created_at":"2025-11-10 16:18:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":207817,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of (a) Chitosan, (b) Carboxy methyl cellulose and (c) Methotrexate.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/0c3ec3391bb649fae6297677.png"},{"id":95566100,"identity":"88b26e00-542f-4618-9d4c-2a960e8effcb","added_by":"auto","created_at":"2025-11-10 16:18:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":561667,"visible":true,"origin":"","legend":"\u003cp\u003e(a, b and c) SEM images of CaCO\u003csub\u003e3\u003c/sub\u003e(CMC) microparticles with different magnifications and (d) XRD pattern of CaCO\u003csub\u003e3\u003c/sub\u003e(CMC) microparticles. In (c), vaterite and calcite are represented by V and C, respectively. SEM images of (e) magnetic multilayer microcapsules and (f) hollow magnetic multilayer microcapsules.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/5606fb94c03d78ef7dec3b93.png"},{"id":95566103,"identity":"a5343213-972b-435c-9112-204a2f2405c7","added_by":"auto","created_at":"2025-11-10 16:18:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":163778,"visible":true,"origin":"","legend":"\u003cp\u003ea) Pore size distribution and (b) plot of the adsorption versus partial pressure of CaCO\u003csub\u003e3\u003c/sub\u003e (CMC) microparticles and (c) pore size distribution of hollow magnetic multilayer microcapsules.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/2659927b172354e856163d49.png"},{"id":95566097,"identity":"531d0cf4-679b-47b2-a5c9-e69e822c3187","added_by":"auto","created_at":"2025-11-10 16:18:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":430465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003ea) TEM image, (b) size distribution and (c) UV-Vis absorption spectrum of Au NPs and (d) SEM image, (e) size distribution, (f) FTIR spectra of the (green) Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e and (yellow) citrate coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/67417034babd95c536729dea.png"},{"id":95654799,"identity":"07189ee2-c02d-4750-8d4f-d8a415409f43","added_by":"auto","created_at":"2025-11-11 16:13:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":459274,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Appearance of microcapsules were changed with incorporation of (1) one layer and (2) two layers of magnetite. The sedimentation of microcapsule suspension was accelerated through use of a magnet (3 and 4); (b) The variation in zeta potential in relation to the number of layers on the surface of calcium carbonate microparticles. (Number 0 represents bare microparticles).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/6711fb6d876cc23debc9c107.png"},{"id":95655673,"identity":"3e0f1ee0-f900-4071-a820-564d5f0bfce6","added_by":"auto","created_at":"2025-11-11 16:16:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":99696,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectra of calcium carbonate microparticles, CS, CMC, (d) hollow magnetic multilayer microcapsules, pure MTX and MTX loaded magnetic multilayer microcapsule.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/7a452e40779ec9a229fa30c7.png"},{"id":95654609,"identity":"3ae26982-ff2a-43b7-8d39-51cea9ac4a1e","added_by":"auto","created_at":"2025-11-11 16:12:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":166418,"visible":true,"origin":"","legend":"\u003cp\u003e(a) UV-Vis absorbance spectra of MTX before (red color) and after (purple color) drug loading process. Inset: alteration in absorbance at the maximum wavelength of MTX (303 nm) is being observed at various incubation durations. (b) Effect of thickness of the layers (MTX concentration was kept 50 ppm), (c) Effect of MTX concentration to microcapsule ratio and (d) Effect of various pH values of microcapsule solution on MTX loading efficiency.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/928bb464a187b4f02008b381.png"},{"id":95655144,"identity":"a1535ed6-1ce6-4829-9c8d-bfb3327d3370","added_by":"auto","created_at":"2025-11-11 16:14:22","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":161766,"visible":true,"origin":"","legend":"\u003cp\u003e(a)In vitro release of MTX from magnetic multilayer microcapsule in different pH, (b) Photothermal effects of water, Au NPs and an aqueous dispersion of magnetic multilayer microcapsule under NIR irradiation (200mW cm\u003csup\u003e−2\u003c/sup\u003e), (c) Release of MTX from magnetic multilayer microcapsule with and without NIR irradiation at 37˚C.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/9651b45817e0815e399821df.png"},{"id":95566107,"identity":"6ac80bc4-f919-44c3-a616-0caf84d8ef12","added_by":"auto","created_at":"2025-11-10 16:18:07","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":57821,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003eCell viability of (a) the drug-free and MTX-loaded magnetic multilayer CS/CMC microcapsules against MCF-7 without/with the assistance of 200mw NIR irradiation for 15 min, after incubation for 24 h, (b)\u003cem\u003e \u003c/em\u003ethe control culture medium without/with 200 mw NIR irradiation for 15 min.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/96b5ebb63df5001c6f9a4224.png"},{"id":100615916,"identity":"18eb43c0-7447-48bb-aec5-f12fc8474784","added_by":"auto","created_at":"2026-01-19 17:38:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3061774,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/9d884358-b6a7-4e50-8485-07ef68eee7d8.pdf"},{"id":95566138,"identity":"babe2d5d-2e7f-446a-ad64-9e241c8cd5bc","added_by":"auto","created_at":"2025-11-10 16:18:08","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":457329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1: \u003c/strong\u003eThe steps of magnetic multilayer microcapsule preparation and MTX loading.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/c065c4cab37d67df4979f925.png"},{"id":95566099,"identity":"44c5e045-e682-4cac-9b8a-129b88191a52","added_by":"auto","created_at":"2025-11-10 16:18:07","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":419405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-7925880/v1/4bdc43a1ce3938e33a0bda88.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of Dual-Responsive Magnetic/NIR-Triggered Multilayer Microcapsules for Controlled Methotrexate Delivery in Cancer Therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer treatment remains a major clinical challenge, as chemotherapy often limited by systemic toxicity, multidrug resistance, and non-specific drug delivery. To overcome these limitations, stimulus-responsive drug delivery systems have emerged as transformative approaches, offering precise, localized drug release triggered by internal or external stimuli such as pH, biomolecules, magnetic fields, or light[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among these, near-infrared (NIR) light-responsive systems are particularly attractive due to their non-invasive application, high tissue penetration depth, and minimal damage to healthy cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Multilayer microcapsules, fabricated via the layer-by-layer (LbL) technique, have demonstrated unique advantages in stimuli-responsive drug delivery owing to their tunable hollow structures, material versatility, and ability to incorporate a variety of functional nanoparticles[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The use of natural polysaccharides and their derivatives in capsule formulation is a promising avenue for drug delivery applications Chitosan (CS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), (1\u0026ndash;4)2-amino 2-deoxy b-D glucan, a polysaccharide abundantly present in crustacean shells, is derived from partially deacetylated chitin. Chitosan (CS) is a widely recognized biopolymer with excellent biocompatibility and biodegradability, making it ideal for use in drug delivery systems [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The polyanionic polysaccharide carboxymethyl cellulose (CMC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) possesses a valuable combination of properties: biocompatibility, biodegradability, and low cost. These characteristics, along with its bioavailability, make CMC a promising candidate for the development of sustainable drug delivery systems[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].Additionally, integrating magnetite (Fe₃O₄) and gold (Au) nanoparticles into the capsule shells allows for dual-functional systems,achieving both magnetic targeting and efficient photothermal response under NIR irradiation[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Au nanoparticles have caused the localized heating to lead to the destruction of microcapsules. This innovative approach has proven effective in releasing encapsulated peptides, proteins, and model drugs, making it a promising technique for precise and targeted drug delivery[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This capability opens up possibilities for remotely controlled drug release and targeted delivery systems[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].Methotrexate (MTX) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), a widely used chemotherapeutic agent, effectively inhibits DNA synthesis but is associated with significant off-target toxicity including leukopenia, hepatotoxicity, nephrotoxicity, and bone marrow suppression[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These side effects significantly limit the effectiveness of the treatment[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] .\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn this study, we propose that magnetic and NIR-responsive multilayer microcapsules constructed from biocompatible CS and CMC polymers can provide an effective platform for MTX delivery. These microcapsules were synthesized via layer-by-layer electrostatic assembly of CS and CMC on a sacrificial CaCO\u003csub\u003e3\u003c/sub\u003e template, incorporating Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e and Au nanoparticles for magnetic targeting and near-infrared (NIR) light-triggered release, respectively. Following EDTA-mediated template removal, the resulting hollow microcapsules were loaded with MTX at various concentrations and pH values. The in vitro cytotoxicity of the MTX-loaded multilayer microcapsules against MCF-7 breast cancer cells was evaluated upon magnetic targeting and NIR irradiation (780 nm). The findings highlight the potential of these microcapsules as advanced platforms for minimally invasive cancer therapy.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eCarboxymethyl cellulose (CMC, M\u003csub\u003ew\u003c/sub\u003e~70 kDa, degree of substitution\u0026thinsp;=\u0026thinsp;0.9), Chitosan (CS, M\u003csub\u003ew\u003c/sub\u003e~10kDa, deacetylation degree: 91%)), Hydrogen tetrachloroaurate trihydrate (HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO), Ferric chloride hexahydrate (FeCl\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO), Ferrous chloride tetrahydrate (FeCl\u003csub\u003e2\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO) and MTT assay (3-[4,5- dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide) were purchased from Sigma Aldrich. Citric acid, Ethylenediaminetetraacetic acid disodium salt (EDTA), Sodium chloride (NaCl), Ammonium hydroxide (NH\u003csub\u003e4\u003c/sub\u003eOH), Calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e), Sodium hydroxide (NaOH), Sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e), were bought from Merck. The MCF-7 human breast cancer cell line (RRID: CVCL_0031) was obtained from the Iranian Biological Resource Center (IBRC), Tehran, Iran. The cells were tested for mycoplasma contamination using a PCR-based Mycoplasma Detection Kit (Pishtaz Teb Diagnostics, Iran) and were confirmed to be mycoplasma-free.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Synthesis of citrate coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles\u003c/h2\u003e\u003cp\u003eAs described previously, the co-precipitation method was used for preparation of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticle (NP) using Fe\u003csup\u003e2+\u003c/sup\u003e and Fe\u003csup\u003e3+\u003c/sup\u003e ions[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In a typical reaction, 25 mL FeCl\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO (0.2 M) and FeCl\u003csub\u003e2\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO (0.1 M) solutions were prepared and mixed at water bath (60\u0026deg;C). The co-precipitation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs was carried out in a three-neck round-bottom flask under nitrogen atmosphere. To initiate the formation process of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs, a solution of ammonium (5 mL of 25% NH\u003csub\u003e3\u003c/sub\u003e) was introduced. Following that, a solution of 0.1g citric acid in 20 mL of water was added, and the temperature was raised to 95\u0026ordm;C. The Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were then exposed to citric acid treatment for 1 hour, resulting in the development of negative charges on their surfaces. The black precipitate was carefully separated through sedimentation using an external magnet, followed by multiple rinses with water to ensure thorough purification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Preparation of Au nanoparticles\u003c/h2\u003e\u003cp\u003eAu NPs were prepared by the protocol outlined by Kimura et al[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To summarize, in 20 mL of deionized water, a solution was prepared by dissolving 0.5 mL of HAuCl\u003csub\u003e4\u003c/sub\u003e (20.8 mmol/l). The resulting faint yellowish solution was then heated to a boiling point. During this process, 1 mL of sodium citrate (0.5%) was added to solution and was vigorously stirred. After 30-minute stirring, yellowish color of the solution was changed to red color, indicating the successful synthesize of Au NPs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Preparation of CaCO\u003csub\u003e3\u003c/sub\u003e microparticles doped with CMC\u003c/h2\u003e\u003cp\u003eCaCO\u003csub\u003e3\u003c/sub\u003e(CMC), also known as calcium carbonate microparticles doped with CMC, were produced following the previously described method [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A solution of Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (100 mL, 0.025 M) was combined with CMC solution (2 mL, 5% w/v). The process began by combining the aforementioned solution with of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (100 mL, 0.025 M) solution at ambient temperature. The CaCO\u003csub\u003e3\u003c/sub\u003e microparticles were obtained by vigorously agitating the precipitate using a magnetic stirrer for a duration of 30 seconds. After sedimentation, they were washed three times with distilled water and subsequently underwent air drying. This resulted in the formation of the CaCO\u003csub\u003e3\u003c/sub\u003e(CMC) hybrid microparticles.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.5. Fabrication of multilayer CS/CMC microcapsules functionalized with Au and Fe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eNPs\u003c/b\u003e.The synthesize of the microcapsules involved a layer-by-layer template-assisted assembly[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The cores were subsequently removed to create the hollow magnetic multilayer CS/CMC microcapsules[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, the microparticles of CaCO\u003csub\u003e3\u003c/sub\u003e(CMC) were dispersed in a solution of CS (2 mg/mL) containing 0.5 M NaCl. The mixture was then gently shaken for a duration of 15 minutes to establish the CS layer on template. Next, the coated microparticles underwent separation through centrifugation (10,000 rpm, 1 min). Subsequently, they were washed three times using a 0.5 M NaCl solution at a pH of 5.0. After being washed with distilled water, the precipitate was dispersed in solution of CMC (1mg/mL) and was shaken for 15 minutes. The microparticles were separated using centrifugation and were subsequently rinsed with a 0.5 M NaCl solution at pH 5.0 for three times. Using a similar method, the negatively charged CMC layer was replaced with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs and Au NPs separately. Then unabsorbed NPs were eliminated through magnetic decantation and washing steps. Consequently, different numbers of bilayers (ranging from 8 to 12) were generated by repeating bilayer coating, while chitosan was selected as the outer shell. The 1% glutaraldehyde (GA) solution was used to cross-link the magnetic multilayer CS/CMC microcapsules at room temperature for a duration of 12 hours [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Hollow microcapsules were prepared by adding EDTA solution (0.2 M at pH 7.0 for 15 min incubation) to removal of template. The hollow microcapsules were subjected to three cycles of rinsing using EDTA, followed by three cycles of rinsing with water. Then the samples were subjected to a drying process in an oven at a temperature of 50\u0026deg;C for 1 hour. Subsequently, they were stored for future investigations. The final shell composition of the microcapsule was (CS/CMC)\u003csub\u003e4\u003c/sub\u003e(CS/Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e(CS/Au NP)\u003csub\u003e2\u003c/sub\u003e(CS/CMC)\u003csub\u003e2\u003c/sub\u003e CS. The steps of multilayer microcapsule preparation and drug loading are drawn schematically (Schem1)\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.6. MTX loading into magnetic multilayer CS/CMC microcapsules\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe preparation of the magnetic multilayer CS/CMC microcapsules discussed in the preceding section was carried out with the exception that, prior to the addition of GA, we introduced magnetic CS/CMC microcapsules (1 mg) into 5 mL MTX solution with different concentrations (10\u0026ndash;200 ppm) and different pH values. The mixture was magnetically stirred at ambient temperature for a duration of 24 hours. MTX\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ewas incorporated into the magnetic CS/CMC microcapsule by physical adsorption. A 0.50 T magnet was used to separate the unloaded MTX from MTX loaded multilayer CS/CMC microcapsules. The concentration of MTX in the supernatant was measured using a UV-Vis spectrophotometer with absorbance at 303 nm. Calibration curve was determined for pure MTX (A\u0026thinsp;=\u0026thinsp;0.0215C\u003csub\u003eppm\u003c/sub\u003e-0.0278) with linear concentration range (5\u0026ndash;50 ppm). The loading efficiency (LE) of MTX was determined using the below equation:\u003c/p\u003e\u003cp\u003eLE (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\mathbf{t}\\mathbf{o}\\mathbf{t}\\mathbf{a}\\mathbf{l}\\:\\mathbf{c}\\mathbf{o}\\mathbf{n}\\mathbf{c}.\\:\\:\\mathbf{o}\\mathbf{f}\\:\\mathbf{d}\\mathbf{r}\\mathbf{u}\\mathbf{g}\\:-\\mathbf{c}\\mathbf{o}\\mathbf{n}\\mathbf{c}.\\:\\:\\mathbf{o}\\mathbf{f}\\:\\mathbf{u}\\mathbf{n}\\mathbf{b}\\mathbf{o}\\mathbf{u}\\mathbf{n}\\mathbf{d}\\mathbf{e}\\mathbf{d}\\:\\mathbf{d}\\mathbf{r}\\mathbf{u}\\mathbf{g}}{\\mathbf{t}\\mathbf{o}\\mathbf{t}\\mathbf{a}\\mathbf{l}\\:\\mathbf{c}\\mathbf{o}\\mathbf{n}\\mathbf{c}.\\:\\:\\mathbf{o}\\mathbf{f}\\:\\mathbf{d}\\mathbf{r}\\mathbf{u}\\mathbf{g}}\\:\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e (Eq.\u0026nbsp;1)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Drug release study\u003c/h2\u003e\u003cp\u003eThe magnetic multilayer CS/CMC microcapsules containing MTX were evenly distributed in a 5 mL buffer solution (0.1M, pH 5.0 and 7.4) and subjected to incubation at 37 \u003csup\u003e◦\u003c/sup\u003eC with gentle agitation. This process was carried out both with and without NIR irradiation, using an output power of 200 mW, at specific time intervals for drug release. The UV-Vis spectrophotometer was utilized to measure the quantity of drug released at 303 nm. The MTX concentration was measured with the help of the standard calibration curve and the cumulative drug release (%) was calculated using the following equation:\u003c/p\u003e\u003cp\u003eCumulative drug release (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\varvec{m}\\varvec{g}\\:\\left(\\varvec{d}\\varvec{r}\\varvec{u}\\varvec{g}\\:\\varvec{r}\\varvec{e}\\varvec{l}\\varvec{e}\\varvec{a}\\varvec{s}\\varvec{e}\\varvec{d}\\right)}{\\varvec{m}\\varvec{g}\\:\\left(\\varvec{t}\\varvec{o}\\varvec{t}\\varvec{a}\\varvec{l}\\:\\varvec{d}\\varvec{r}\\varvec{u}\\varvec{g}\\right)}\\:\\times\\:100\\:\\:\\)\u003c/span\u003e\u003c/span\u003e (Eq.\u0026nbsp;2)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Cytotoxicity investigation\u003c/h2\u003e\u003cp\u003eFor cytotoxicity investigation, human breast cancer MCF-7 cell line was used. The cell line was initially cultured in 96 well microplate and were kept in a humid atmosphere (95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e) at 37\u0026deg;C and the medium was refreshed. After this time, the cells were incubated in medium supplemented with different concentrations of MTX and To regulate the crystallization and growth patterns of the CaCO3 microparticles throughout the preparation procedure, researchers introduced carboxymethyl cellulose (CMC) into the reaction mixture. CMC is a polysaccharide with a negative charge, which was incorporated to achieve the formation of spherical microparticles of CaCO3. This addition of CMC helped in controlling the shape and size of the synthesized microparticles. Additionally, the presence of CMC in the reaction mixture has the added benefit of preserving the biocompatibility of the synthesized microparticles. synthesized hollow microcapsules (10\u0026ndash;40 ppm) for 48 h. During the photothermal treatments, a NIR light with an intensity of 200mW was used to irradiate the cells for 15min. After a 48-hour incubation period, MTT (5mg/mL in phosphate buffered saline (PBS)) was introduced to each well and allowed to incubate for 4 h at 37\u003csup\u003e◦\u003c/sup\u003eC. To disperse the formazan crystals, the supernatant containing MTT was extracted and subsequently, 100 \u0026micro;l of dimethyl sulfoxide (DMSO) was introduced. The plate was subsequently stored at ambient temperature for a duration of 30 minutes. The absorbance of the solution was determined at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varvec{\\lambda\\:}=570\\:\\mathbf{n}\\mathbf{m}\\)\u003c/span\u003e\u003c/span\u003e using a plate reader (Awareness), with background subtraction performed at\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\varvec{\\lambda\\:}=630\\:\\mathbf{n}\\mathbf{m}\\)\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Characterization\u003c/h2\u003e\u003cp\u003eThe Philips instrument, Model XL-30, was utilized to conduct SEM analysis (accelerating voltage of 26 kV). The TEM images were captured by employing an FEI TECNAI transmission electron microscope (accelerating voltage of 120 kV). Particle size distributions of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e and Au NPs and the zeta-potential of the CaCO\u003csub\u003e3\u003c/sub\u003e(CMC) microparticles after each layer coating was determined by a Zeta-sizer (Malvern Instruments Ltd.). The CaCO\u003csub\u003e3\u003c/sub\u003e(CMC) microparticles were analyzed using the Bernauer-Emmett-Teller technique to determine their specific surface area and pore size distribution. The surface analyzer model (BEL Japan) was utilized to conduct this measurement via N\u003csub\u003e2\u003c/sub\u003e adsorption desorption at -196 \u003csup\u003e◦\u003c/sup\u003eC. X-ray diffractometer (Philips) equipped with Cu Kα radiation were used to record the XRD patterns. FT-IR spectrometer (Bruker, ALPHA, Germany) in the range of 4000\u0026ndash;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was used to characterize the structure of synthesized materials. UV-Vis spectrophotometer (Agilent 8453) was utilized to record the absorption spectra in the UV-visible range. A laser beam operating at a wavelength of 780 nm in the near infrared (NIR) range, with a power output of 200 mW was utilized.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Synthesize and characterization of magnetic multilayer CS/CMC microcapsule\u003c/h2\u003e\u003cp\u003eThe SEM micrographs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and b) reveal that the CaCO\u003csub\u003e3\u003c/sub\u003e(CMC) microparticles possess a regular spherical structure with approximately 500 nm in size. Furthermore, the SEM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) provide evidence of their porous morphology. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, the XRD pattern reveals that the microparticles of CaCO\u003csub\u003e3\u003c/sub\u003e(CMC) primarily consisted of vaterite, with a small presence of calcite [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The presence of carboxymethyl cellulose (CMC) in preparation of CaCO3 microparticles hindered the recrystallization process[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Hence, the inclusion of carboxymethyl cellulose (CMC) in the CaCO\u003csub\u003e3\u003c/sub\u003e microparticles significantly enhanced their stability compared to the particles without any additives[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The pore size distribution of CaCO\u003csub\u003e3\u003c/sub\u003e (CMC) was analyzed using the BJH method, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. The results revealed that the average pore diameter of the microparticles was 5.6 nm. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb presents additional evidence from nitrogen adsorption-desorption measurements, showing a significant increase in surface area, rising from 8.8 m\u0026sup2;/g for CaCO\u003csub\u003e3\u003c/sub\u003e microparticles without CMC to 128.4 m\u0026sup2;/g.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This indicates that the presence of CMC greatly enhances the surface area and porosity of the CaCO\u003csub\u003e3\u003c/sub\u003e microparticles. The adsorption of CMC polymer molecules onto the carbonate surface causes a loosening of the microparticles' packing, resulting in an increase in surface area.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo enhance the properties of CaCO\u003csub\u003e3\u003c/sub\u003e microparticles, a coating consisting of 10 bilayers was applied. The coating comprised of alternating layers of chitosan (CS) with a positive charge and sodium carboxymethyl cellulose (CMC), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, and Au NPs with negative charge. This multi-layer coating aimed to improve the functionality and performance of the microparticles to achieve high charges for both CS and CMC, the polymer solutions were adjusted to an optimal pH of 5.0 using 1.0 M NaOH or HCl. This pH value was chosen as it ensures that both CS and CMC exhibit a high level of charge[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Au NPs were incorporated into microcapsules for the purpose of thermal therapy application. The characterization of the Au NPs using TEM and DLS techniques revealed that they possess a diameter ranging from 6 to 10 nm. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). The Au NPs displayed the peak absorption at 560 nm in the UV-Vis spectrum, as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec. According to zeta potential measurements, the carboxyl groups of sodium citrate result in a negative surface charge of the Au NPs, exhibiting a zeta potential of -23.5 mV.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlso, in order to achieve remote control and precise manipulation of microcapsules towards the desired location, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were synthesized and incorporated as a middle layer of multilayer microcapsules. As shown in the scanning electron microscopy (SEM) image (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs exhibited a spherical morphology, possessing an average diameter ranging from 50 to 80 nm. The size of NPs was further verified with DLS analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). To verify that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e was successfully prepared and coated with citrate, FTIR spectra of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e and citrate coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). In citric acid, the peak at 1715 cm\u003csup\u003e-1\u003c/sup\u003e, which corresponds to the C\u0026thinsp;=\u0026thinsp;O vibration, shifts to 1618 cm\u003csup\u003e-1\u003c/sup\u003e when citric acid is coated onto the magnetite surface. The Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs exhibited a zeta potential of -18.3 mV upon measurement, indicating that the negative surface charge of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs is generated by carboxyl groups of sodium citrate. With introducing Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs into microcapsules, their appearance was changed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea (1) and (2)) and their density increased; resulting in more rapid sedimentation from a he utilization of a magnet in the process can further expedite the sedimentation of microcapsules, resulting in a faster settling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea (3) and (4)). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, when the zeta potential was measured at pH 5.0 for different coating steps, a reversal of the surface charge from positive to negative was observed. The adsorption of polyelectrolytes onto the surface is confirmed by this reversal of zeta potential. Since CMC is negatively charged, the obtained microparticles exhibited a zeta potential of -23.0 mV. The presence of alternating positive and negative charge potential variations on the surface of the capsules indicates that the polycation CS and polyanion CMC have been successfully coated on the CaCO\u003csub\u003e3\u003c/sub\u003e(CMC) microparticles in an alternating manner[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Finally, the CS outer layer of multilayer microcapsules was cross-linked by glutaraldehyde. Previous studies have shown that glutaraldehyde undergoes a reaction with materials that contain primary amine groups, such as chitosan, certain peptides, and polypeptides [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The result of this reaction is the formation of covalently cross-linked polymers. After the required number of layers were loaded, the template was removed using ethylenediaminetetraacetic acid (EDTA), resulting the hollow multilayer microcapsules. As shown in SEM image (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), the prepared multilayer microcapsules showed a spherical shape and smooth surface morphology. The addition of different layers of polymers onto the porous CaCO\u003csub\u003e3\u003c/sub\u003e(CMC) microparticles transforms the rough surface into a smoother one. This is attributed to the slight agglomeration that occurs between the oppositely charged polyelectrolytes, which act as bridges between each other, promoting adhesion. The SEM image of the hollow multilayer CS/CMC microcapsule (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) showed CaCO\u003csub\u003e3\u003c/sub\u003e template has been completely removed. BET analysis of the hollow multilayer microcapsules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) reveals a mean pore diameter of 3.6 nm, with a porosity of approximately 42%. To analyze the magnetic multilayer CS/CMC microcapsules, we employed FT-IR spectroscopy for characterization purposes. IR spectra of CaCO\u003csub\u003e3\u003c/sub\u003e microparticles, CS, CMC, magnetic multilayer CS/CMC microcapsules are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The spectral analysis of the CaCO\u003csub\u003e3\u003c/sub\u003e microparticles shows significant absorption peaks at 1380 cm\u003csup\u003e-1\u003c/sup\u003e, 1010 cm\u003csup\u003e-1\u003c/sup\u003e, 877 cm\u003csup\u003e-1\u003c/sup\u003e, and 746 cm\u003csup\u003e-1\u003c/sup\u003e. These peaks are characteristic modes of vibration for carbonate[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].With respect to CS spectrum, the N-H and O-H stretching vibration is responsible for the distinctive absorption band observed at 3450 cm\u003csup\u003e-1\u003c/sup\u003e and the peaks at 1649 and 1603 cm\u003csup\u003e-1\u003c/sup\u003e are attributed to the amide I and II bands[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In the spectrum of CMC), the O\u0026ndash;H stretching vibration of the OH group is responsible for a wide band observed at 3419cm\u003csup\u003e-1\u003c/sup\u003e, while the C\u0026ndash;H stretching vibration of the CH\u003csub\u003e2\u003c/sub\u003e and CH\u003csub\u003e3\u003c/sub\u003e groups is indicated by the band at 2920 cm\u003csup\u003e-1\u003c/sup\u003e.The COO group can be associated with the asymmetric and stretching vibrations, which are indicated by the absorption peaks observed at 1616 cm\u003csup\u003e-1\u003c/sup\u003e and 1423 cm\u003csup\u003e-1\u003c/sup\u003e, respectively[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. For magnetic multilayer CMC/CS microcapsules, compared to the spectra of CS and CMC, The characteristic bands of -NH\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and -COO\u003csup\u003e-\u003c/sup\u003e, which represent the absorption of N-H bonds of primary amine group and COO group respectively, were observed at 1412 and 1597 cm\u003csup\u003e-1\u003c/sup\u003e. These findings indicate the presence of a strong electrostatic interaction between the carboxylic acid salts (-COO\u003csup\u003e-\u003c/sup\u003e) and the amino groups (-NH\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) within the microcapsule[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The distinguishing characteristic of the magnetic multilayer CMC/CS microcapsules' spectrum is the prominent peak observed at 593 cm\u003csup\u003e-1\u003c/sup\u003e, which correspond to the vibration of the Fe-O bond.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2. MTX loading studies\u003c/h2\u003e\u003cp\u003eBy simple incubation of the hollow microcapsule in MTX solution, the MTX was loaded into the magnetic multilayer CS/CMC microcapsule. The observed phenomenon of a smoother surface on the coated porous CaCO\u003csub\u003e3\u003c/sub\u003e(CMC) microparticles can be attributed to the strong interaction between the carboxyl and amino groups of drugs with the CS and CMC polymers.\u003c/p\u003e\u003cp\u003eThis interaction leads to the formation of bridges between the oppositely charged layers, resulting in the agglomeration of the different layers and a smoother surface. To investigate the interaction between MTX and magnetic multilayer CS/CMC microcapsules, the FTIR spectra of magnetic multilayer CS/CMC microcapsules were examined both before and after MTX loading. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e, for MTX, the main peaks of COOH at 1693cm\u003csup\u003e-1\u003c/sup\u003e, -CONH at 1643cm\u003csup\u003e-1\u003c/sup\u003e, aryl system at 1543cm\u003csup\u003e-1\u003c/sup\u003e and 1500cm\u003csup\u003e-1\u003c/sup\u003e and aromatic ring system at 830cm\u003csup\u003e-1\u003c/sup\u003e are present. The N-H group is indicated by the peak at 3380 cm\u003csup\u003e-1\u003c/sup\u003e. The stretching vibrations of the C-C or C-H bond are represented by peaks at 1490 and 1209 cm\u003csup\u003e-1\u003c/sup\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The spectral group frequencies of MTX-loaded magnetic multilayer CS/CMC microcapsules (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e) were characterized at 2507cm\u003csup\u003e-1\u003c/sup\u003e and 2928cm\u003csup\u003e-1\u003c/sup\u003e for C-H vibrations. The MTX-loaded microcapsules exhibit a higher wavenumber (3430 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for the N-H stretching peak, as different with free MTX (3380 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). This observation indicates an interaction between the NH\u003csub\u003e2\u003c/sub\u003e group of free MTX and the COOH group of the microcapsule, leading to a shift in the peak. The vibration absorptions at 1643 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e attributed to amide of free MTX shifted to a higher wavenumber (1673 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e, further confirms the interaction of COOH and NH\u003csub\u003e2\u003c/sub\u003e groups. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ea compares the UV-Vis absorption intensities of the DOX peak at 303 nm before and after the drug-loading process. Various factors influencing the drug loading capacity are examined in the following sections.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1. The impact of layer thickness on loading efficiency of MTX\u003c/h2\u003e\u003cp\u003eIn this work, we investigated the effect of layer thickness on the loading efficiency of MTX. The thickness of the layers was identified as a crucial factor in determining the loading efficiency of the drug. By examining the relationship between layer thickness and drug loading, we aimed to gain insights into optimizing the loading process and enhancing the effectiveness of MTX delivery. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eb exhibits that the number of coated layers have impact on the MTX loading efficiency (The MTX concentration remained constant at 50 ppm). The loading efficiency of MTX was maximum and 42% when 10 bilayers were applied onto the multilayer microcapsules' surface. The study found that as the number of layers increases (up to 10), the drug loading efficiency does not change and remains constant. This indicates that 10 bilayers are the ideal choice for synthesizing microcapsules with high loading efficiency.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2. The impact of different concentrations of MTX on loading efficiency\u003c/h2\u003e\u003cp\u003eTo investigate the impact of different concentrations of MTX on drug loading efficiency, the magnetic CS/CMC microcapsules were combined with MTX solution of varying concentrations. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, the maximum loading efficiency of MTX into microcapsules is determined 68% at concentration of 90 ppm. This finding suggests that the drug loading efficiency is greatly influenced by the concentration of MTX. At higher feeding concentration of MTX, the loading efficiency was decreased because of insufficient functional groups in the polymer concentration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3. The impact of different pH values on loading efficiency\u003c/h2\u003e\u003cp\u003eThe MTX loading efficiencies of magnetic CS/CMC multilayer microcapsule solutions were evaluated across a pH range of 2.0 to 6.0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). The synthesized magnetic multilayer CS/CMC microcapsule exhibited a greater drug loading efficiency at pH 5.0 thus we select this pH for further experiments.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3. NIR- and pH-simulated MTX release from the magnetic multilayer CS/CMC microcapsules\u003c/h2\u003e\u003cp\u003eThe investigation of the release of MTX from the magnetic multilayer CS/CMC microcapsules was conducted under physiological conditions (37\u0026ordm;C) with and without NIR irradiation. The experiments for release were carried out at pH levels of 5.0 and 7.4, considering the fact that tumor tissue has acidic pH levels, whereas normal tissue maintains a pH of around 7.4. This allowed the researchers to assess how the microcapsules respond to different pH conditions and evaluate the potential for targeted drug delivery in tumor tissues.\u003c/p\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1. \u003cem\u003eIn vitro\u003c/em\u003e drug release under different pH values\u003c/h2\u003e\u003cp\u003eDrug release rate from magnetic multilayer CS/CMC microcapsule was slow and sustained to more than 35 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). The drug release profiles of carrier were compared at pH 5.0 and pH 7.4. The results are obviously indicated that drug release at an acidic pH of 5.0 was much faster than physiological pH, emphasizing that drug release from microcapsule is controlled by pH, meaning the smaller toxicity of the MTX loaded magnetic multilayer microcapsule to normal tissues (pH 7.4). This represents advantages of a safe system for drug delivery.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2. Drug release investigation under NIR irradiation\u003c/h2\u003e\u003cp\u003eAu NPs have been employed as agents for photothermal conversion in the field of photothermal therapy due to their high near-infrared (NIR) light absorption coefficients [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb indicate the photothermal effects of water, Au NPs and magnetic multilayer CS/CMC microcapsule under NIR irradiation (200mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). The photothermal effects of these materials can be summarized as follows: water does not exhibit significant photothermal effects under NIR irradiation, as it has a low absorption coefficient for NIR light. Au NPs exhibit efficient photothermal heating capabilities when exposed to NIR light. Due to presence of Au NPs, the magnetic multilayer CS/CMC microcapsule display photothermal properties that can be influenced by the presence of NIR light. The Au NPs and multilayer CS/CMC microcapsules show promising photothermal properties, making them suitable for applications in cancer treatment and other medical fields. These findings indicated that the produced heat caused by Au NPs is sufficient to fulfill the requirement for enhancing near-infrared (NIR) responsive drug release[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. When irradiation time is increased, the drug release increases (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). After approximately 15 min of irradiation, up to 85% of the loaded MTX was released, while less than 7% was released when incubated in the dark for the same duration. Additionally, the temperature increase followed a consistent pattern, with a rapid initial rise and then reaching a plateau after approximately 40 minutes of exposure to irradiation. The release of MTX from the microcapsules is triggered by the temperature increase and collapse of the microcapsules resulting from the photothermal effect of Au NPs upon NIR irradiation. The study findings suggest that a duration of 15 minutes of irradiation is sufficient for the complete rupture of the capsules and the subsequent release of the drug. Also, in our work, a cross-linking agent called GA was introduced to increase the stability of the multilayer coating on the template using the LbL technique. However, GA is not able to withstand high temperatures. When the Au NP within the microcapsule shells were exposed to NIR light, the temperature inside the shells increased significantly. As a result, the aldehyde groups found in GA was degraded, leading to the instability and degradation of the multilayers of microcapsule[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Consequently, the loaded MTX was liberated from the microcapsules. The responsiveness of our system to external near-infrared (NIR) stimulus surpasses that of simple diffusion-based release systems. This finding highlights the significant potential of our system as a drug delivery system that is both highly responsive and easily controllable.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.4. MTT assay\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea shows the cell viability of the bare and MTX-loaded magnetic multilayer CS/CMC microcapsules in the concentration range of 10 to 40 ppm without/with the irradiation of 200 mw NIR for 15 min. Clearly, without NIR irradiation, the drug-free magnetic multilayer showed low cytotoxicity against the MCF-7 cancer cells, in while with 15 min of NIR irradiation, the toxicity of drug-free magnetic multilayer CS/CMC microcapsules significantly enhanced and 32\u0026ndash;65% of tumor cells were killed in the concentration range of 10 to 40 ppm.\u003c/p\u003e\u003cp\u003eHowever, the cytotoxicity remained largely unaffected in the control experiment (with and without NIR irradiation) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb), suggesting that the presence of magnetic multilayer CS/CMC microcapsules is essential for the observation of the photothermal effect. This result emphasizes that the prepared magnetic multilayer CS/CMC microcapsules have the potential to serve as a highly effective photothermal agent for eradicating tumor cells when exposed to NIR irradiation. Under irradiation of NIR, the prepared magnetic multilayer CS/CMC microcapsules can serve as a highly effective photothermal agent, capable of effectively eliminating tumor cells. Their unique properties make them ideal for targeted photothermal therapy in various biomedical applications. For MTX-loaded magnetic multilayer CS/CMC microcapsules, without the assistance of NIR irradiation, the cell viability only reduced by 88\u0026ndash;65% as the concentration increased from 5 to 40 ppm, which is understandable because of a little release of the loaded MTX the first 24 h. In contrast, with NIR irradiation for 15 min, for the MTX-loaded magnetic multilayer microcapsules in the same concentration range, cell viabilities decreased from 50 to 16%. A significant increase in the cytotoxicity of MTX-loaded magnetic multilayer CS/CMC microcapsules against cancer cells can be seen under 15 min of NIR radiation that indicates the developed magnetic multilayer CS/CMC microcapsules could be used in both chemo- and photothermal treatment, leading to a highly effective therapeutic outcome. This combined approach offers great potential for achieving excellent therapeutic efficacy in various biomedical applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study successfully fabricated novel biocompatible hollow magnetic multilayer CS/CMC microcapsules incorporating Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e and Au NPs by LbL technique. These microcapsules exhibited precise magnetic guidance and demonstrated high sensitivity to NIR irradiation, leading to a significant enhancement in drug release compared to diffusion-based systems. From the cellular cytotoxicity study, under NIR irradiation, MTX-loaded magnetic multilayer CS/CMC microcapsules showed higher toxicity than cytotoxicity of drug-free magnetic multilayer CS/CMC microcapsules. This platform, combining magnetic targeting, NIR-triggered drug release, and photothermal therapy capabilities, holds significant promise for advancing targeted cancer treatment and other biomedical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThis work is based upon research funded by Iran National Science\u003cbr\u003e\u0026nbsp;Foundation (INSF) under project No. 4033973.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available in the main manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT author statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMojtaba Shamsipur:\u003c/strong\u003e Conceptualization,\u0026nbsp;Validation,\u0026nbsp;Resources,\u0026nbsp;Writing - Review \u0026amp; Editing,\u0026nbsp;Supervision,\u0026nbsp;Project administration,\u0026nbsp;Funding acquisition\u003cstrong\u003e\u0026nbsp;Samira Kariminia:\u0026nbsp;\u003c/strong\u003eMethodology,\u0026nbsp;Software,\u0026nbsp;Formal analysis,\u0026nbsp;Investigation,\u0026nbsp;Data Curation,\u0026nbsp;Writing - Original Draft,\u0026nbsp;Visualization\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCybulla E, Vindigni A (2023) Leveraging the replication stress response to optimize cancer therapy. 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Int J Biol Macromol 2023:128215\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section\u003c/p\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Drug delivery, Multilayer microcapsule. Responsive drug release, NIR irradiation","lastPublishedDoi":"10.21203/rs.3.rs-7925880/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7925880/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNear-infrared (NIR)-responsive drug delivery systems offer precise spatiotemporal control for targeted cancer therapy with minimal invasiveness. Here, we report the design of magnetic/NIR-responsive multilayer microcapsules via layer-by-layer assembly of biocompatible chitosan (CS) and carboxymethyl cellulose (CMC). Incorporation of magnetite (Fe₃O₄) and gold nanoparticles (Au NPs) into the microcapsule shells enabled both magnetic guidance and efficient NIR-triggered drug release. Methotrexate (MTX), a potent anti-cancer agent, was encapsulated with a high loading efficiency (68%). Systematic studies revealed the influence of bilayer number, MTX concentration, and environmental pH on drug loading and release kinetics. \u003cem\u003eIn vitro\u003c/em\u003e drug release was studied in different pH values and in the presence of an NIR irradiation. While the drug releases much faster in an acidic environment (pH 5.0) compared to physiological pH (7.4), it maintains stable release across both conditions, leading to uniform therapeutic delivery. Under NIR irradiation for15 min, up to 85% of the loaded MTX was released, while less than 7% was released when incubated in the dark for the same duration, significantly enhancing cytotoxicity against MCF-7 breast cancer cells. These dual-responsive microcapsules offer a versatile platform for targeted chemotherapy and photothermal therapy, highlighting their potential in minimally invasive cancer treatment.\u003c/p\u003e","manuscriptTitle":"Development of Dual-Responsive Magnetic/NIR-Triggered Multilayer Microcapsules for Controlled Methotrexate Delivery in Cancer Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 16:18:02","doi":"10.21203/rs.3.rs-7925880/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-20T10:42:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-12T07:07:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-04T13:32:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-03T02:23:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-31T02:37:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74194372782486729349860994109209813067","date":"2025-10-30T13:07:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146828536671874452156012156636110323164","date":"2025-10-30T01:20:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65707471109405543476469075176110354411","date":"2025-10-30T00:28:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9953523655908602115430288694208728824","date":"2025-10-29T13:22:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-29T13:15:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-23T05:55:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-23T05:54:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Polymers and the Environment","date":"2025-10-22T17:32:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f2000202-1523-4fd2-8cd1-65963a1586f5","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T17:02:35+00:00","versionOfRecord":{"articleIdentity":"rs-7925880","link":"https://doi.org/10.1007/s10924-025-03728-9","journal":{"identity":"journal-of-polymers-and-the-environment","isVorOnly":false,"title":"Journal of Polymers and the Environment"},"publishedOn":"2026-01-12 16:29:02","publishedOnDateReadable":"January 12th, 2026"},"versionCreatedAt":"2025-11-10 16:18:02","video":"","vorDoi":"10.1007/s10924-025-03728-9","vorDoiUrl":"https://doi.org/10.1007/s10924-025-03728-9","workflowStages":[]},"version":"v1","identity":"rs-7925880","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7925880","identity":"rs-7925880","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}