Preparation of MnO2@poly-(DMAEMA-co-IA)-conjugated methotrexate nano-complex for MRI and radiotherapy of breast cancer application

A novel efficient pH-sensitive targeted magnetic resonance imaging (MRI) contrast agent and innovative radio-sensitizing system were synthesized based on MnO2 NPs coated with biocompatible poly-dimethyl-amino-ethyl methacrylate-Co-itaconic acid, (DMAEMA-Co-IA) and targeted with methotrexate (MTX). The as-established NPs were fully characterized and evaluated for MRI signal enhancement, relaxivity, in vitro cell targeting, cell toxicity, blood compatibility, and radiotherapy (RT) efficacy. The targeted NPs MnO2@Poly(DMAEMA-Co-IA) and MTX-loaded NPs inhibited MCF-7 cell viability more effectively than free MTX after 24 and 48 h, respectively, with no noticeable toxicity. Additionally, the insignificant hemolytic activity demonstrated their proper hemo-compatibility. T1-weighted magnetic resonance imaging was used to distinguish the differential uptake of the produced MnO2@Poly(DMAEMA-Co-IA)-MTX NPs in malignant cells compared to normal ones in the presence of high and low MTX receptor cells (MCF-7 and MCF-10A, respectively). In MRI, the produced theranostic NPs displayed pH-responsive contrast enhancement. As shown by in vitro assays, treatment of cells with MnO2@Poly(DMAEMA-Co-IA)-MTX NPs prior to radiotherapy in hypoxic conditions significantly enhanced therapeutic efficacy. We draw the conclusion that using MnO2@Poly(DMAEMA-Co-IA)-MTX NPs in MR imaging and combination radiotherapy may be a successful method for imaging and radiation therapy of hypoxia cells.


Introduction
Cancer is a confusing and frightening disease and the second most common cause of death worldwide, is characterized by uncontrolled cell proliferation and lack of cell death, which eventually leads to the formation of an abnormal tumor mass.This cancerous condition has led researchers to develop a variety of techniques for the rapid and accurate diagnosis and treatment of cancer [1,2].Molecular imaging is a rapidly developing field and crucial for cancer imaging through shifting diagnostic focus from dysfunction that appears later in a disease process to the biochemical events which precipitate the early disease stage [3].Molecular magnetic resonance imaging (MMRI) offers the possibility of imaging several events at the cellular and intracellular levels.Many significant advances have been made recently in this field due to the ability to generate images with non-invasive, non-ionizing radiation, and high spatial resolution and proper soft tissue contrast [4].Since any molecular imaging application needs a particular imaging probe, new chemical approaches become progressively important.Because they can detect variations and changes in molecular composition, MRI contrast agents (CAs) are crucial in the development of MRI for molecular imaging [5].
Advances in nanoparticle technology over the last decade have shown that some of these materials may play a prominent role in molecular imaging and cancer therapy [6,7].In MRI imaging, prolonged loosening of water protons leads to a weak difference between different tissues, leading in a poor contrast of normal and malignant tissue.In order to address this challenge, CAs can be employed to improve the contrast in MRI.To enhance image contrast, various T 1 -or T 2 -MRI CAs based on paramagnetic complexes, such as gadolinium (Gd), manganese (Mn), and iron oxide nanoparticles (Fe 3 O 4 NPs), have been created [8,9].Gd 3+ -based T 1 CAs are most widely applied in clinical applications because Gd 3+ ion has seven unpaired electrons that can effectively reduce T 1 levels.Gd 3+ -based CAs have short circulation times and their use is limited due to free Gd 3+ ions that might be dissociated from chelates, which are responsible for nephrogenic systemic fibrosis (NSF) in patients [10].
Negative contrast agents like clinically approved superparamagnetic iron oxide (SPIO) nano-CA, which provide high transverse relaxation (T 2 ) and decrease the signal-tonoise ratio (SNR) as well as produce negative contrast in T 2 -weighted MRI images [11].Compared to paramagnetic T 1 CA that provides bright contrast and enhance the signal intensity, the dark regions formed by the signal decreasing effect of superparamagnetic T 2 agents could be confused with other pathogenic conditions, such as bleeding, calcinations, etc.Moreover, high magnetic susceptibility of the T 2 CA induces distortion of the magnetic field on neighboring tissues, leading to the creation of blooming artifacts in which situation lesions appear larger than they actually are.Therefore, the development of ''positive'' contrast agents for cell imaging could provide signal enhancement effects and can be easily distinguished from other pathogenic or biological conditions and hence T 1 CAs are widely used in the clinics [12,13].
Another prospect for CAs for MRI that may be able to overcome some of the disadvantages of gadolinium is manganese (Mn)-based CAs.There are two basic causes for this.Since manganese is a naturally occurring element in the human body and plays a significant role in the metabolism of lipids, carbohydrates, and proteins, cells including hepatocytes, cardiomyocytes, and pancreatic tissue quickly absorb free ions [14,15].Therefore, the presence of an endogenous elimination pathway might be a great advantage but, however, determined Mn concentration levels must be respected.And also, Mn shows all the Gd physical properties: high spin quantum number (five unpaired electrons in its 3D orbit), long longitudinal electronic relaxation times, and faster water exchange kinetics.Moreover, differently than Gd, Mn has a low T 2 at high field strength MRI that make it useful also in T 2 -weighted imaging [16].
So, Mn-based CAs with fewer side effects can be considered as an appropriate substitute for Gd compounds in T 1 -weighted MRI images [17].Chelators were utilized in this instance as well, though, to make more stable probes and shelter Mn from cellular uptake.Due to their high T 1 relaxivity and potential to have a therapeutic effect through the release of therapeutic medicines or as a result of external stimuli, the manganic dioxide and oxide nanoparticles have also received a lot of interest [18].
Manganese dioxide (MnO 2 ) NPs have many outstanding properties, which are expected to make further progress in cancer detection and treatment.Under a redox reaction that MnO 2 does with GSH, which overexpressed GSH in cancer cell media, it produces Mn +2 , which increases MRI contrast.Healthy and tumor cells differ mainly in three aspects: hypoxia, acidosis, and excessive H 2 O 2 .The invasion of tumors, their ability to spread, and their resistance to treatment are a result of hypoxia in the tumor [19].
Therefore, it is crucial during treatment to minimize hypoxia at the tumor location.Several methods are now being used to address hypoxia, including increasing blood flow to the tumor site to improve oxygen levels, delivering oxygen to tumor locations via oxygen carriers (such as perfluorocarbons), and reducing oxygen consumption at tumor sites [20].However, due to biological safety, each of these approaches has some limits.For this reason, specific nanocomplexes with high catalytic capacity, ideal size and structure, and higher stability were developed [21].A catalase-like enzyme found in MnO 2 NPs can interact with extra H 2 O 2 at tumor regions to treat tumor hypoxia.Additionally, MnO 2 consumes hydrogen protons at the tumor site and is converted to water-soluble Mn +2 , which can be eliminated by the kidneys and be employed as a contrast agent for MR imaging.These special MnO 2 NPs have been extensively utilized to lower tumor hypoxia due to their special qualities [22].Targeting drug delivery has long been a problem for medical researchers who are trying to get drugs to the right place in the body and how to control drug release to prevent overdose.Therefore, it is highly desirable to develop a stimulus-responsive drug delivery system capable of delivering the drug to the desired site [23].Methotrexate (MTX) anticancer drug, as an anionically charged and a stoichiometric inhibitor of dihydro-folate reductase, is clinically applied for the treatment of human malignancies [24].However, its clinical efficacy is hampered by unwanted side effects due to its low permeability and non-specific drug delivery.Additionally, the folatemediated endocytosis response of MTX provides the benefits of active targeting of NPs, resulting in new formulations with low carrier-induced toxicity, enabling strong and efficient cellspecific uptake [25].
In light of the above information, the goal of this project was to improve and design a targeted biocompatible nanocomplex to be applied as an MRI CA and radiotherapy sensitizer.In this regard, MnO 2 NPs were synthesized and coated with itaconic acid (IA) and bound to MTX.The MnO 2 core NPs in this nanostructure are dissociated into Mn +2 , which enhances T 1 magnetic resonance signals in acidic media and is also a well-known RT sensitizer that may cause endogenous H 2 O 2 in the tumor microenvironment to decompose, producing oxygen, and overcoming RT resistance associated with hypoxia.Due to the high specific surface area achieved by MnO 2 NPs and the hydrophilicity of the IA layer, high accessibility of water molecules to the MnO 2 core and consequent acceptable relaxivity would be expected, which is basically beneficial in enhancing MR imaging signal.MTX loading capacity and release profile, in vitro cytotoxicity of the prepared samples against cancerous cells (MCF-7) and normal cells (MCF-10A), and blood-compatibility of the samples were entirely evaluated.In vitro MRI experiment was also evaluated to study the potential of the developed targeted nano-system in MRI.The MnO 2 component has the capacity to efficiently and quickly promote the decomposition of H 2 O 2 into O 2 , thereby overcoming the intrinsic hypoxic environment of tumors and, more importantly, rendering our treatment approach independent of O 2 .

Synthesis of MnO 2 NPs
MnO 2 NPs were synthesized by a modified procedure described in the literature with some modifications [26].
In order to create a dark purple solution, 0.7 g of potassium permanganate (KMnO 4 ) was first dispersed in 10 mL of deionized water for 5 min.The mixture was then given 0.8 g of NaoH to add, which was then stirred for 24 h at room temperature until the solution turned brown.Finally, the mixture was centrifuged (800 rpm) and the supernatant was discarded and washed three times with deionized water to eliminate any residual permanganate.It was finally dried in a desiccator and MnO 2 NPs were obtained in a spherical structure.

Synthesis of poly-(dimethylaminoethyl methacrylate-co-itaconic acid) poly-(DMAEMA-Co-IA)
Azobisisobutyronitrile (AIBN) was used as the radical initiator during the copolymerization of DMAEMA and IA, which took place in DMSO at 70 °C.[DMAEMA]/[IA]/ [AIBN] had a molar ratio of 85:15:100.DMAEMA (0.3 g, 1.9 mmol), IA (0.4 g, 3.1 mmol), and AIBN (0.034 g, 0.2 mmol) were typically dissolved in dry DMSO in order to make poly(DMAEMA-Co-IA).Nitrogen bubbles were used to degas the reaction system for 30 min.The polymerization reaction was then carried out for 24 h at a temperature of 70 °C in a nitrogen environment with constant mechanical stirring.The excess diethyl ether was used to precipitate the polymer solution, which was then filtered and vacuum dried.

Preparation of MnO 2 @Poly(DMAEMA-Co-IA)
In this regard, 10 mg MnO 2 and 10 mg of IA were mixed in 10 mL distilled water and stirred for 48 h at room temperature.Then, the unreacted polymers were removed by centrifugation at 6000 rpm for 10 min.The pellet was redistributed in distilled water for further use.

Conjugation of MTX to MnO 2 @Poly(DMAEMA-Co-IA)
A first, 20 mg of the prepared MnO 2 @Poly(DMAEMA-Co-IA) was dissolved in 5 mL distilled water and stirred properly until the solution became homogeneous.Afterward, 200 mL MTX (50 mg/mL) was added to the medium and stirred for another 48 h at room temperature.

Evaluation the amount of MTX loading in MnO 2 @ Poly(DMAEMA-Co-IA)
First, the prepared MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs were centrifuged at 4000 rpm for 10 min and the supernatant was collected.In order to define MTX loading capacity and efficiency, an indirect technique in which the unloaded MTX in the supernatant was calculated using a MTX standard calibration curve experimentally via ultraviolet-visible (UV-Vis) spectroscopy at a detection wavelength of 300 nm.MTX loading efficiency as well as capacity were determined according to the below equations [27].

Characterization of nano-complex
A transmission electron microscopy (TEM) image was taken using a Hitachi H7650 transmission electron microscope and an acceleration voltage of 120 kV.An X'Pert PRO MPD PANalytical (the Netherlands) X-ray diffractometer with a Cu target was employed to collect the X-ray diffraction (XRD) patterns (40 kV, 40 mA).The Fourier-Transform Infrared (FTIR) spectra were obtained using the KBr technique at room temperature with an FTIR spectrometer from Bruker Corp in Germany.For the purpose of determining the zeta potential and hydrodynamic size distribution of the samples in deionized water at room temperature, Dynamic Light Scattering (DLS) analysis was carried out using the Nicomp 380 ZLS Zeta Potential/Particle Sizer (PSS Nicomp, USA).Utilizing induced plasma atomic emission spectroscopy, the amount of Mn was determined (ICP-OES 730-ES, Varian).The magnetic characteristics of the samples were assessed using Vibrating Sample Magnetometer (VSM) measurements with a maximum magnetic field of 10 kOe at 25 °C using VSM Model 7400 (VSM, Meghnatis Kavir Kashan Co., Kashan, Iran).A 1.50 T MRI scanner was utilized to perform the in vitro MR imaging (Siemens Prisma MRI scanner using a head coil).

Relaxivity measurements
To evaluate the generated samples suitability as an MR contrast agent, the longitudinal (T 1 ) relaxation time of the MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs was determined using spin-echo sequence by applying 1.5 T MRI system equipped with the head coil.According to the ICP results, multiple concentrations of MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs (0.64, 0.32, 0.16, 0.08, and 0.04 mM) were made by diluting the original sample solution in distilled water at various pH values in phosphate buffer (pH 7.4), acetate buffer, and buffer solutions (pH 6.5).Some amounts of H 2 O 2 were added to the phantom environment to simulate hypoxia conditions.Dotarem was utilized at the same Mn concentration as a control, and all samples were dispersed in 2% (w/v) agarose gel.T 1 -weighted images were obtained with following parameters: external field (H) = 1.5 T, temperature = 22 °C, NEX = 3, slice thickness = 2 mm, (1) Loading Capacity (%) = Mass of loaded MTX Mass of final product × 100 flip angle of 90°, number of signal averages = 3, field of view = 128 × 128 mm 2 , 256 × 256 matrix size, and bandwidth = 15.63Hz/Px.The various repetition times (TR) and the fixed echo time (TE) values were utilized as TR/ TE = 50, 200, 400, 600, 800, 1100, 1300, 1500, 1800, and 2000/11 ms.To acquire T 1 relaxation times, these methods were carried out for five concentrations of the NPs.The reverse relaxation time (1/T 1 ) as a function of NPs concentration was fitted and the r 1 relaxivity was obtained from the slope of related curves.The identical method was applied to measure the r 2 relaxivity for prepared NPs and Dotarem.The T 2 -weighted images were obtained using a multispin-echo sequence with the similar parameters as T 1 -weighted images acquisition except for TR/TE = 3000/10, 30, 60, 90, 130, 170, 210, 240, 270, and 350 ms, slice thickness = 5 mm.

In vitro MR imaging
To test the targeting ability of the MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs, MCF-7 cells with high MTX receptor expression, 4T 1 and MCF-10A (cancerous and normal cells with negligible MTX receptors) were incubated for 6 h at 37 °C with different concentrations of the prepared NPs (0, 1, and 50 µgr per mL).MR imaging was performed after the cells had been incubated, washed in PBS, and re-suspended in PBS at a cell density of 1 × 10 6 cells/mL.To get MR images, a 1.50 T Siemens Prisma MRI equipment was employed.The standard spin-echo procedure was used to acquire T 1 -weighted MR images with the following setting: TR/TE = 500/12 ms, 220 × 320 matrix, 82 × 120 mm 2 field of view (FOV), 140 Hz/Px bandwidth and slice thickness of 3 mm.As a final point, R 1 (1/T 1 ) values were evaluated by a mono-exponential fitting algorithm [28].

Cytotoxicity assay
Using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, the cytotoxicity of the MnO 2 @ Poly(DMAEMA-Co-IA) NPs, free MTX, and MnO 2 @ Poly(DMAEMA-Co-IA)-MTX NPs against breast normal and cancer cells was assessed (MCF-10A and MCF-7, respectively).MCF-10A cells were selected to evaluate the cytotoxicity effect because normal tissues are naturally acidic, but the microenvironment of malignant tissues and intracellular organelles including endosomes and lysosomes is mildly acidic [29].Separately planted in 96-well plates with 5 × 10 3 cells per well in 200 L of RPMI and DMEM media, respectively, normal and malignant cells were then incubated for 24 h.The old medium was then replaced with new media containing 200 µL of MnO 2 @Poly(DMAEMA-Co-IA) NPs, MTX, and MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs at various concentrations (0-100 µg/mL) in a total volume of 200 µL.The plates were then incubated at 37 °C for an additional 24 and 48 h before each well received 100 µL of PBS containing 0.5 mg/mL of MTT.The cells were then treated with 100 µL of DMSO following an additional 4 h of incubation.The absorbance of each well was measured using a Bio Tek microplate reader (USA) at a wavelength of 570 nm, and cell viability was determined as follows: The final test findings were expressed as the mean value and standard error of the mean of three independent experiments performed in triplicate.

Hemolysis assay
According to the earlier literature, human red blood cells (HRBCs) were produced for hemolytic assay.Ethylenediamine-tetra-acetic acid (EDTA) was used to fix fresh human blood cells, which were then centrifuged for 5 min at 2000 rpm.The precipitate was then cleaned with PBS (pH 7.4).PBS was used to dilute the HRBC suspension 10 times.Then, 800 mL of each sample was added separately to 200 mL of the suspension at various concentrations (0-2000 µg/mL), and the results were contrasted with controls that were both positive (800 mL Triton X100, 2% v/v) and negative (800 mL PBS, pH = 7.4).Then, 2 h of room temperature incubation and moderate shaking were carried out.The absorbance of hemoglobin in the supernatants was then measured using a UV-Vis spectrophotometer at 541 nm.The hemolytic activity (in percentage) was acquired using the subsequent formula: Standard deviations (SD) were acquired from the triplicates.

Hypoxia cell culture
The concentration of oxygen in the normal air is 21% (paO 2 = 160 mmHg).Inside the body, the partial pressure of oxygen in the blood decreases significantly from the arteries to the tissue cells, so that in pulmonary vessels: paO 2 is 80-100 mm Hg, in arteries: 40-60 mm Hg, and in cells: 1-20 mm Hg.Each tissue and cell has a different ability to adapt to these new conditions.An important question in biological research is how cells can adapt to low O 2 concentrations (hypoxia) that occur in cells and organs during inflammation [29].In this investigation, N 2 gas was utilized to simulate a hypoxic situation.Therefore, before adding the MnO 2 @Poly(DMAEMA-Co-IA)-MTX complex, N 2 gas was purged in PBS solution for 20 min to eliminate any dissolved oxygen [30].

Cell irradiation
Siemens ONCOR Linear Accelerator Megavoltage (MV) X-ray beams were used to irradiate MCF-7 cells (Siemens AG, Henkestr, Erlangen, Germany).Radiation beams of 6 MV were utilized to deliver doses of 0, 2, 4, and 8 Gy to the samples at a rate of 300 MU/min.The treated cell plate should be surrounded by water-filled cell plates that have a field size of 17*13 cm 2 and a source axis distance (SAD) of 100 cm [31].This will ensure that all cells get uniform radiation treatment.Additionally, the cell plates were positioned near the beam's center on top of a 5 cm block of polystyrene with enough accumulation to generate enough backscatter.Following radiation, the irradiated and control cells were put back in the incubator.

Apoptosis assay
Flow cytometry assay was performed to define the cell death for treated and untreated MCF-7 cells.In brief, after cultivation of 4 × 10 5 cells per well in a 6-well plate, breast cancer cells were treated with 50 µg/mL dose of MnO 2 @ Poly(DMAEMA-Co-IA) NPs and MTX for 24 h.Additionally, radiation groups were exposed to 8 Gy of absorbed radiation from 6 MV radiation beams (at a dosage rate of 300 MU/min.The following step involved staining the collected cells with Annexin V-FITC and Propidium Iodide (IQ products, the Netherlands).After 15 min, the ratio of apoptotic cells was defined by a flow cytometer (Partec CyFlow ® ) [32].

Statistical analysis
Every data item was presented as mean ± standard error.The statistical differences between groups were examined via one-way ANOVA (random effect).The p value limit for statistical significance was less than 0.05.

Characterization of MnO 2 @ Poly(DMAEMA-Co-IA)-MTX NPs
The pH-sensitive and adsorbing portion of the MnO 2 surface was selected as PIA.The pH-sensitive section for MTX conjugation was chosen to be PDMAEMA.This is accomplished by creating the biocompatible copolymer Poly(DMAEMA-Co-IA) and conjugating MTX, a targeting molecule, in order to enhance the uptake of NPs into cancer cells.The direct chemisorption of carboxylic groups completes the interaction between MnO 2 and MTX-conjugated Poly(DMAEMA-Co-IA) in an aqueous medium.Figure 1 shows the steps involved in making MTX-conjugated poly(DMAEMA-Co-IA).Some authors hypothesized that the (-COO-) of PIA would interact with MnO 2 NPs, and that the residual carboxylate groups would enhance the repulsion effect, resulting in more stable NPs in aqueous media [33].When compared to some related studies, the drug loading content and encapsulation efficiency of the MTX in the NPs structure were determined to be 33% and 58%, respectively.
It is evident that all of the MnO 2 @Poly(DMAEMA-Co-IA) NPs diffraction peaks fit perfectly with typical MnO 2 , and no additional impurity peaks could be seen.The existence of an amorphous coating layer on the surface of MnO 2 may be the cause of the modest intensity decrease in the MnO 2 @ Poly(DMAEMA-Co-IA) NPs XRD pattern when compared to pure MnO 2 .

Size and Zeta potential
Zeta potential (surface charge) measurements serve as an indirect indicator of the NPs physical stability.The shape, average size, and size distribution of NPs can all be used to determine their stability.Additionally, the homogeneity of the size distribution is typically measured using the polydispersity index (PDI).

FTIR analysis
The FTIR spectrum of MnO 2 NPs was shown in Fig. 3.The presence of the Mn-O bond within the MnO 2 structure is shown by the prominent absorption band at 502 cm −1 , which can be attributed to the Mn-O stretching mode.While the absorption band at 2857 cm −1 results from a very different degree of hydrogen bonding within the sample, the absorption bands at 1724.32 cm −1 , 1554 cm −1 , 1419.91 cm −1 , and 1100.81cm −1 match to the O-H bending vibrations linked with Mn atoms.The existence of absorbed water molecules within the MnO 2 structure is indicated by the discovery of O-H vibrations in the FTIR spectrum [35][36][37].
The spectrum of poly-(DMAEMA-co-IA) displays broad -OH stretching absorption peaks in the 2800-3600 cm −1 range, a strong and narrower -C=O stretching peak at approximately 1659 cm −1 corresponding to carboxylic acid, a peak at 1406 cm −1 corresponding to C-O-H in-plane bending, and a peak at 1214 cm −1 corresponding to C-O stretching for PIA segment [38].Moreover, PDMAEMA displayed peaks at 2943 cm −1 , and 1450 cm −1 corresponding to methylene group, carboxylic C=O stretch and -OH bending, respectively [39].The overlap of primary amine and hydroxyl groups was identified by the FTIR spectrum analysis as a wide band at 3344 cm −1 .Symmetric and asymmetric CH 2 stretching vibrations were found to be the causes of the weak shoulder absorption at 2948 cm −1 .
The FTIR spectrum revealed all of the characteristic peaks of poly-(DMAEMA-co-IA) and MnO 2 , indicating that the synthesis of MnO 2 @Poly(DMAEMA-Co-IA) NPs was successful.The spectrum of MTX-loaded MnO 2 @ Poly(DMAEMA-Co-IA) NPs showed a slight shift in position in the characteristic bands due to the formation of hydrogen bonds between the MTX and the hydroxyl and amino groups in poly-(DMAEMA-co-IA).

Morphology and structure of MnO 2 @ Poly(DMAEMA-Co-IA) NPs
The size of NPs was evaluated by electron microscopy.TEM analysis of the NPs specified that they were approximately spherical in shape.Figure 4 showed TEM images of MnO 2 NPs with sizes extending from 20 to 30 nm in diameter.Magnetic hysteresis (M-H) curves (field dependency of magnetization) of MnO 2 NPs and MnO 2 @Poly(DMAEMA-Co-IA)-MTX nanocomposite recorded by VSM at room temperature in a maximum field of 10 kOe (Fig. 5).The M-H curves showed that MnO 2 NPs and MnO 2 @Poly(DMAEMA-Co-IA)-MTX nanocomposite have modest ferromagnetic properties; however, the ferromagnetic hysteresis loop could not be seen for either sample.As shown in Fig. 5, the saturation magnetization (Ms) of the MnO 2 @Poly(IA-PDMAEMA)-MTX nanocomposite is lower than that of the MnO 2 NPs, which may be because the produced polymers have decorated the MnO 2 NPs.MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs have weaker magnetic properties than bare MnO 2 NPs due to poorer contact between the intercalated MnO 2 NPs and the co-polymer sheets than bare MnO 2 NPs.Furthermore, neither sample demonstrates saturation remnant magnetization (Mr) or coercive force (Hc) [40].
Colloidal stability of MnO 2 @ Poly(DMAEMA-Co-IA)-MTX NPs DLS analysis was used to determine changes in the hydrodynamic size of NPs in various media, including DI water, PBS (pH = 7.4), and DMEM supplemented with FBS (10%), in order to evaluate the colloidal stability of the final synthesized NPs.MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs in PBS displayed adequate stability by exhibiting no significant size variation over a period of 7 days [Fig.6].This might be the result of the media creating an equilibrium between attractive and repellent interactions [41,42].The size of the NPs in FBS/DMEM medium (10%) did not change noticeably at the same time.This might be explained by the BSA protein adsorption to the NPs, increasing their stability [43].
In other words, BSA serves as an auxiliary component giving the NPs steric stability.According to the DLS findings, the NPs were more stable in PBS and FBS/DMEM (10%) media, respectively, due to electrostatic repulsion and steric repulsion [44].However, their aggregation or flocculation, which led to a shift in their hydrodynamic diameter, revealed the NPs poor stability in DI water.The results illustrated a trend toward an increasing in hydrodynamic diameter, even if not statistically significant.The outcomes validated the produced NPs colloidal stability as being acceptable for additional biological applications.

Hemolysis assay
All substances that enter the blood are in contact with red blood cells.Therefore, in order to evaluate the effect of different concentrations of NPs on Red blood cells (RBCs), the hemo-compatibility analysis using hemoglobin release measurement spectrophotometry has been recognized as one of the most important experiments to assess the biosafety of NPs on blood erythrocytes.The results showed that the extent of hemolysis is a concentration-dependent process and the percentage of red blood cells hemolysis increases with an increasing concentration of drug-carrying NPs.As shown in Fig. 7b, the percentage of erythrocytes lysis activity of drug-carrying NPs in the investigated concentration range (1.95-2000 μg/mL) was appropriate and less than 5% (standard acceptance limit) indicated the suitable blood compatibility of the prepared NPs [11].

Cytotoxicity
The results of MTT analysis for MTX, MnO 2 @ Poly(DMAEMA-Co-IA) NPs, and MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs against MCF-10A and MCF-7 cell lines after 24 and 48 h of incubation time have been indicated in the Fig. 8.The lower toxicity of MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs in normal cells compared to cancerous cells can be a result of the slow and controlled release of the MTX result in appropriate toxicity on healthy cells and tissues.
The in vitro toxicity of MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs was assessed against MCF-10A normal cells with low expression of MTX receptors, and MCF-7 malignant cells exhibiting over-expression of MTX receptors to take into account the targeting capacity of the created NPs [45].At 24 and 48 h, the cell viability assays were conducted.MnO 2 @Poly(DMAEMA-Co-IA) NPs minimal cytotoxicity toward both cell types (in the absence of an anticancer drug) suggests that the carrier is biocompatible (Fig. 8).After 48 h, the lowest cell viability was greater than 90% at the highest concentration (100 µg/mL).With the lengthening of the incubation period and increased MTX content, the viability of both cell lines fell off drastically.Following a 48 h treatment with drug-loaded NPs at a concentration of 100 µgr/mL, as shown in Fig. 8, cell viability for MCF-7 and MCF-10A cells was around 40% and 45%, respectively, while these values for free MTX NPs were approximately 44.8% and 60.2%, respectively (p < 0.05).This increased cellular absorption of MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs than free MTX may be a result of the system's targeting ability, which allows for more penetration through lipid cell walls and the selective accumulation of MTX in malignant cells [46].Furthermore, at the highest concentration, MnO 2 @ Poly(DMAEMA-Co-IA)-MTX NPs were significantly less toxic to MCF-10A (a normal cell line) (about 62.3% after 24 h and 68.2% after 48 h) than MCF-7 (a cancer cell line) (p < 0.05).This may be attributed to the overexpression of MTX receptor on the surface of cancerous cells (active targeting), which caused targeted cell uptake through the mechanism of receptor-mediated endocytosis, leading to increased accumulation of the MTX anticancer medication in cancerous cells compared to normal cells.Additionally, tumor cells produce anticancer drugs at a higher rate in an acidic environment than they do in a natural environment.

Relaxivity measurement
T 1 -weighted images from the NPs were somewhat brighter than the commercially approved MRI contrast agent (Dotarem) in phantom model (pH = 6.5).In comparison to Dotarem, it was revealed that MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs effectively reduced the longitudinal relaxation time (T 1 ) and greatly increased signal intensity in T 1 -weighted images.However, it was observed that the amount of the bright contrast increase in T 1 -weighted images for all groups was correlated with the Mn ion concentration.(IA-PDMAEMA) NPs groups was not statistically significant (p = 0.066).Also, the concentrations of both MTX and MnO 2 @(IA-PDMAEMA)-MTX NPs ≤ 12/5 µg/ml have no significant effect on cell viability of MCF-7 and MCF-10A for incubation times of 24 and 48 h (p > 0.05).However, higher concentrations significantly decreased the viability of the cells for these groups (p < 0.05).It was found that the combination of MTX and MnO 2 @(IA-PDMAEMA) NPs showed a statistically significant effect in both cell lines and meaningfully decreased the cell viability (p < 0.001) compared to cells treated only with MnO 2 @ (IA-PDMAEMA) NPs.Besides, no significant difference was shown between MTX and MnO 2 @(IA-PDMAEMA) groups At a lower pH, the r 1 relaxivity values for MnO 2 @ Poly(DMAEMA-Co-IA)-MTX NPs were 5.52 mM −1 s −1 .This result could be due to the structure of the coating layer, which causes more water molecules to be absorbed into the nanoparticle structure, leading to an increase in their longitudinal relaxation.There is no significant difference between the relaxivity of Dotarem in different pH media (2.39 and 2.48 mM −1 s −1 , respectively).
In the acidic environment, MnO 2 @Poly-(DMAEMA-Co-MR IA)-MTX signal intensities noticeably decreased with an increase in Mn concentration, demonstrating their T 2 -weighted response in lower pH media.Again, there is no significant difference between the relaxivity of Dotarem in various pH media (3.87 and 3.75 mM −1 s −1 , respectively).
The r 2 /r 1 ratio value (1.28) showed that the T 1 -shortening effect in MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs was dominant over the T 2 effect, indicating the strong contrast enhancement in T1-weighted images of the produced nano-system.
In the crystallized structure of MnO 2 , Mn +4 is bonded to six O 2-atoms to form a mixture of edge and cornersharing MnO 2 .In this structure, Mn +4 ions have 3 unpaired electrons in their d orbital (3d 3 ).But at lower pH values, Mn +4 becomes Mn +2 , which has five unpaired electrons in the 3d orbital (3d 5 ).Therefore, the MRI signal is at lower pH where Mn +2 ions are present in the structure, and have a higher MRI signal intensity than the normal pH value for Mn +4 .In Dotarem (Gd-DOTA) molecule, the oxidation number of Gd +3 is 3 and has 7 unpaired electrons in its f orbital (4f 7 ), which has MRI signal and no matter whether it is in acid medium or in natural medium.
While MnO 2 is known to be stable at neutral pH levels, it would break down into Mn +2 and O 2 at lower pH levels according to the following chemical reaction.In order to have the following chemical reaction conditions and the effect of the acidity of the environment, the pH of the environment was lowered using acetate buffer and some amounts of H 2 O 2 were added to the environment [47].Mn +2 is a great T 1 -shortening agent in MR imaging and has five unpaired 3D electrons.
Moreover, the tight coordination between the manganese ions and oxygen atoms in MnO 2 NPs provides the manganese ions with a spatial shield against water molecules [48].As a result, they are unable to induce the relaxation of protons spin-lattice or spin-spin.However, Mn +2 , a high T 1 -weighted contrast agent, may be effectively released when they are in contact with the acidic environment [49].
In order to explore that whether the environment pH value could trigger the dissolution of the prepared NPs into enough Mn +2 , for T 1 -weighted imaging, MRI of NPs in acid and natural media was assessed.
The results demonstrated that MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs failed to exhibit the satisfactory imaging effect at pH 7.4, as there is no noticeable difference between different concentrations of MnO 2 @Poly(DMAEMA-Co-IA)-MTX.However, in an acidic environment, MnO 2 @ Poly-(DMAEMA-Co-MR IA)-MTX signal intensities considerably increased with an increase in Mn concentration, indicating their prospective usage as a potential T 1 contrast agent in response to the pH environment.The enhanced whiting phenomenon may be due to the different medium, which was also reported by Rohrer et al. [50].It should be noted that the MRI of MnO 2 @Poly(DMAEMA-Co-IA)-MTX under acid environment was noticeably stronger than that of Dotarem as a commercially available CA molecule at the same medium and no matter whether it is in acid medium or in natural medium.
According to the relaxivity analysis, our produced NPs have a stronger T 1 -shortening effect and are practical to employ as positive MRI contrast agents because of their higher r 1 value and prominently displayed concentrationdependent whitening effect.Consequently, the created nanosystem based on MnO 2 NPs could provide pH-triggered MRI CA because MnO 2 dissolution in an acidic environment was capable of producing enough manganese Mn +2 ions for T 1 -weighted imaging.Therefore, it was worth a try to apply it for detecting tumor because of the acidic microenvironment of the tumor tissue.
In this study, Dotarem was used as control group as a commercially available CA for T 1 relaxation time in numerous studies.Various r 1 have reported for Dotarem from 0.75 to 4.89 mM −1 s −1 according to experiments conditions [51].The reduction of positive contrast enhancement of Dotarem in comparison with prepared NPs could be due to the extracellular performance of this standard contrast agent.
In a study conducted by Sebyung Kang et al. in 2021 on a targeting ligand switchable Gd (III)-DOTA/protein cage nanoparticle, it was shown that Gd (III)-DOTA-AaLS/ HER-2Afb and Gd (III)-DOTA-AaLS/EGFR2Afb exhibited high ( 5) r 1 relaxivity values of 57 and 25 mM −1 s −1 at 1.4 and 7 T, respectively, which were ten-fold or higher than those of the clinically applied Dotarem [52].
In the study by Rohrer et al., magnetic properties of MRI contrast media solutions at different magnetic field strengths were compared and relaxivity for gadoterate on 1.5 T has been described as r 1 = 2.9 mM −1 s −1 and r 2 = 3.2 mM −1 s −1 [53].
The r 1 and r 2 values depend on various factors, such as the type of NPs, particle, and hydrodynamic diameter and the properties of ligands or coating agents surrounding the paramagnetic ions, magnetic field value, and the pH, and more studies need to be performed to find the optimum coating layer thickness and particle size of the target CA [54,55].
The similar results were demonstrated in the study by Wenwen et al., as well as in which multifunctional chlorine e6 (Ce 6 ) loaded MnO 2 NPs with surface polyethylene glycol (PEG) modification (Ce 6 @MnO-PEG) were produced [56].

Cellular MR imaging
The presence of Mn +2 in the created MnO 2 @ Poly(DMAEMA-Co-IA)-MTX NPs provided the capability for improved T 1 -based metabolic imaging.According to Fig. 10, compared to MCF-10A normal cells, the relaxation rate (R 1 ) of MCF-7 cells treated with MnO 2 @ Poly(DMAEMA-Co-IA)-MTX NPs was significantly greater.Because cancer cells have a lower pH and a more acidic internal environment than normal cells, this elevated R 1 value in MCF-7 cells may be attributed to the accumulation of paramagnetic Mn +2 that was induced by MnO 2 NPs.While MnO 2 is known to be stable at neutral and basic pH levels, it would break down into Mn +2 and O 2 at lower pH levels [56].
Since Mn +2 is a great T 1 -shortening agent in MR imaging and has five unpaired 3D electrons, in T 1 -weighted MR images of MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs samples at reduced pH medium of MCF-7 cells, a clear concentration-dependent brightening effect was seen, but the signals of the developed NPs at natural pH 7.4 of MCF-10A cells seemed to be somewhat weaker.Since the tumor microenvironment is acidic, MnO 2 @Poly(DMAEMA-Co-IA)-MTX nano-carriers may act as a pH-responsive and potential cellular and metabolic MR imaging agent.This makes them particularly valuable for tumor imaging.
After 6 h of treatment with the nanoparticles at the same concentrations in 4T 1 cells with hardly detectable MTX receptor expression, the T 1 -weighted MR images and the relaxation rate (R 1 ) of the cells were recorded.This was done in order to support the MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs ability to target cancer cells in MR imaging as well as their specific ability to bind to and be taken up by MTX receptors.The R 1 value of MCF-7 cells was three times greater than that of 4T 1 cells after incubation with the MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs (10 µg/mL) for 6 h, as further demonstrated by a quantitative measurement of the R 1 change, as shown in Fig. 10.The significant difference between MCF-7 cells and 4T 1 cells in MR imaging could be attributed to the considerable difference between cellular uptake of the MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs into MCF-7 cells due to over-expressing MTX receptor compare to negligible MTX receptors expression in 4T 1 cells.

Cell irradiation
Figure 11 depicts the findings on the viability of MCF-7 cells when exposed to various concentrations of MTX, MnO 2 NPs, and MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs before and after 6 MV (2, 4, or 8 Gy) radiation doses.It is clear that at 8 Gy, the radiation dose increased in a way that caused the most cell damage and increased cellar damage.
Cell viability was equal to 92% without the NPs at 8 Gy, but when MnO 2 NPs were added to the cell at doses of 0, 12.5, 25, 50, and 100 µg/mL, the viability was reduced to 83.56%, 74.9%, 68.3%, 66.3%, and 53.3%, respectively.Additionally, Fig. 11 showed the survivability of the cells for concentrations of 0, 12.5, 25, 50, and 100 µg/mL of MTX following an 8 Gy radiation dosage.As can be observed, several cell viability values were obtained, including 78.9%, 71.7%, 68.7%, 56.7%, 41.1%, and 33.3%.Cell viability was equal to 77.6%, 69.8 T, 64.7%, 53%, 33.2%, and 29.4%, respectively, for treated cells with MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs at radiation dose of 8 Gy.The data shown in Fig. 10 actually indicate that cellular damage increases as radiation exposure does.Additionally, it demonstrated how varying MTX concentrations affected cell damage.As can be shown, as MTX concentration increased from 0 to 100 µg/mL, cell viability decreased from 66.36 to 29.2% at the highest concentration at radiation dose of 8 Gy.Fu et al. produced HA-MnO 2 NPs able to bind to CD44 receptors overexpressed in glioma cells and, at the same time, thanks to the response of MnO 2 to the tumor acid environment, produce O 2 reducing tumor hypoxia [57].
It could be concluded that the hypoxia-associated radiotherapy resistance could be overcome by the proposed MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs by reacting with endogenous tumor H 2 O 2 to create O 2 [58].the radio-sensitivity of gold nano-spikes (GNS) through hyperthermia in the combined treatment of cancer photothermal (PTT) by near infrared radiation (NIR).Quantitative results obtained by flow cytometry showed that the presence of GNS, NIR and GNS + NIR mainly increased cell apoptosis but did not increase necrosis.The level of apoptosis was increased by NIR PTT due to the presence of GNS.In addition, the synergistic treatment of GNS + NIR + X-ray resulted in the highest total apoptosis ratio of 17.92% [59].

Apoptosis assay
Their results are consistent with the present study.In summary, cell apoptosis and necrosis assays showed that more cell death caused by NPs combined with laser irradiation can be attributed to cell apoptosis and necrosis, especially for cell apoptosis.

Conclusions
In summary, a multifunctional theranostic nano-platform has been successfully designed through the Itaconic Acid-DMAEMA surface conjugation of MnO 2 NPs with MTX for controlled drug administration for targeted therapy and MRI contrast-enhanced imaging.The obtained MnO 2 @ Poly(DMAEMA-Co-IA)-MTX NPs could react with H 2 O 2 in tumor sites to release Mn 2+ for enhancing T 1 -weighted MR imaging, and with endogenous tumor H 2 O 2 to generate O 2 and overcome hypoxia-associated RT resistance in order to improve the efficacy of radiation treatment.Our results indicated that the released Mn 2+ ions from MnO 2 core NPs in tumor environments, induce an ultrasensitive pH responsiveness.T 1 -weighted MRI contrast agents of the developed NPs enable tumor-specific positive contrast enhancement of MR images as compared to widely used contrast agents in clinical MRI as well as efficient loading capacity of chemotherapeutic drugs, whereas both manganese oxide and MTX shell increase radiation therapy efficiency.In vitro studies showed that NPs had significantly improved longitudinal relaxivity, making it easier for MRI to identify the tumor precise position and shape.The produced NPs was taken up specifically by MCF-7 cancer cells as opposed to normal MCF-10A cells due to the cancer cells' over-expression of the MTX receptor on their surface.The composite agent demonstrated outstanding radio-sensitizer therapeutic potential and caused the release of MTX under radiation, producing a precise and synergistic therapeutic impact of radiation therapy for cancer.It was discovered that MTX and radiation therapy together were more cytotoxic than either therapy alone.
In addition, manganese-based contrast agents for MRI have recently attracted attention.These agents were carefully planned to complex manganese in a way that simultaneously gives excellent relaxivity and resistance to manganese dissociation.Manganese's potential application in conjunction with nanotechnologies is being researched.Mn-DPDP (Teslascan, DPDP = dipyridoxyl di-phosphate) was the only FDA approved manganese-based CAs as a liver imaging agent.This CA had a safety factor that was 5 times higher than Gd-DTPA.However, it was withdrawn due to its poor clinical performance and toxicity, as a consequence of Mn-DPDP dephosphorylation and simultaneous transmetallation with zinc in the blood, In conclusion, even though in vivo research is definitely necessary, the findings of the in vitro experiments indicated that the developed targeted theranostic system would be a promising biocompatible candidate for tumor imaging and radiotherapy.

Fig. 9 a
Fig. 9 a T 1 -weighted and b T 2 -weighted MR images of MnO 2 @ Poly(DMAEMA-Co-IA)-MTX nanoparticles and Dotarem as a clinically available contrast agent with various concentrations in water phantom.Prior to MR imaging, the samples were incubated at two Annexin V-FITC/PI was used to assess the apoptosis rate of MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs with and without using radiation in MCF-7 cell line.The results confirmed that the treatment of MCF-7 cells with MnO 2 @ Poly(DMAEMA-Co-IA)-MTX NPs particularly increased cell death compared to the un-treated group (p < 0.05).As illustrated in Fig.12, concentration of 50 µg/mL enhanced apoptotic rate in negative control, cells treated with Radiation as positive control, cells treated with MnO 2 @ Poly(DMAEMA-Co-IA) NPs, cells treated and MnO 2 @ Poly(DMAEMA-Co-IA) NPs with Radiation, cells treated with MTX, cells treated with MTX and Radiation, cells treated with MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs and cells treated with MnO 2 @Poly(DMAEMA-Co-IA)-MTX plus Radiation groups.As shown in Fig.12, gamma radiation alone did not increase the rate of cell apoptosis compared to the control group.It was also found that MTX combined with NPs slightly increased the amount of apoptosis compared to the control group.Apoptosis induction was significantly increased in the [NPs + radiation] group.As can be seen, the combination [NPs + MTX + radiation] resulted in the highest level of apoptosis.The apoptosis rate is 0.14% 22.41%, 31.82%,73.7%, 27.78%, 74%, 74.1%, 89.4% for each group respectively.In a study,Ma et al., investigated

Fig. 10 a
Fig. 10 a T 1 -weighted MR images of MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs in MCF7, MCF-10A and 4T 1 cells at different concentration of NPs after incubation for 6 h on 1.5 T MR system.b R 1 (1/ T 1 ) analysis for T 1 -weighted images at different NPs concentration in MCF7, MCF-10A and 4T 1 cells media

Fig. 11
Fig. 11 Cell viability experiment by various concentrations of MnO 2 nanoparticles (NPs) (A), MTX (B) and MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs (C) on MCF-7 cell line after exposure to 6 MV (2, 4, 8 Gy) radiation.The amount of NPs in a sample is indicated on the x-axis (µg/mL).The average value obtained from three samples is displayed in each bar.Statistical significance is denoted by asterisks.(Student's t test; *p < 0.05 and **p < 0.01) Table1depicts the average size, PDI, and zeta potential.MnO 2 @Poly(DMAEMA-Co-IA) NPs had an average size of 87.60 ± 2.4 nm and a PDI of 0.221, while MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs had a size distribution of 92.7 ± 1.0 nm and a PDI of 0.203.Furthermore, zeta potential measurements were performed on MnO 2 @ Poly(DMAEMA-Co-IA) and MnO 2 @Poly(DMAEMA-Co-IA)-MTX NPs, yielding zeta potential values of 31.6 ± 4.1 and 25.9 ± 3.2 mV, respectively.A significant number of protonated amino groups in the poly(DMAEMA-Co-IA) copolymer produce a positively charged surface.Due to the strong electrostatic repulsive forces between the NPs, the greater value of the zeta potential suggests that the nanoparticle may exhibit good stability in aqueous solutions.The MnO 2 -based nanocomposite showed an average PDI range of between 0.20 and 0.22.