Introduction
Nanoparticles, with their unique physical and chemical traits, offer a promising way to overcome the limitations of traditional therapies for life-threatening diseases like cancer [1,2]. Their ability to be designed for targeted therapy is a key advantage, as it allows for more precise delivery of therapeutics to the diseased sites. This precision is crucial in cancer therapy, as it can minimize the toxic effects on healthy tissues [3,4]. Systems based on inorganic metal nanoparticles offer a wide range of surface modifications, which can be utilized for imaging and targeting through molecular interactions, thereby enhancing their theranostic potential [5-7]. Magnetic nanoparticles (MNPs) are a fascinating type of nanomaterial that has been broadly researched for numerous technological applications [8-10]. In biomedical fields, MNPs are employed to produce heat for hyperthermia treatments and facilitate the remote management of targeted payload delivery, including serving as magnetic resonance imaging (MRI) contrast agents (CAs) [11-14]. Among magnetic materials, iron oxide NPs have received significant interest in the field of nanomedicine, which are obtained in three natural forms such as α-Fe2O3 (hematite), γ-Fe2O3 (maghemite), and Fe3O4 (magnetite) [15,16]. These various kinds of iron oxide NPs demonstrate several encouraging characteristics, such as biocompatibility, minimal toxicity, decreased susceptibility to oxidation, enhanced stability in magnetic behaviour, the potential to transfer superparamagnetic state through reduced particle size, easy synthesis, and surface modifications. Superparamagnetic NPs offer high potential in various applications due to their distinctive physicochemical, thermal, and mechanical properties [17,18]. Many drug formulations utilizing iron oxide NPs have received permission from the Food and Drug Administration (FDA) for commercial use, such as Ferinject®, Venofer®, and Ferrlecit® for treating iron-deficient anaemia [19].
MRI offers state-of-the-art imaging solutions to radiologists and clinicians for proper diagnosis based on visualizing tissue morphology and anatomical features in both humans and animals. While MRI is a prominent imaging technique in oncology, its sensitivity is limited in the context of molecular and cellular imaging [20-22]. Magnetic contrast agents are often used to improve contrast and strengthen signals. While gadolinium diethylenetriaminopentaacetic acid (Gd-DTPA) is commonly used in clinical practice due to its strong T1 shortening effect, it has weak contrast enhancement and a rapid retention time in vivo [23,24]. Furthermore, the toxicity and biological safety of gadolinium during and after cellular endocytosis and its deposition in the brain remain largely unclear. Recent research indicates that magnetic iron oxide NPs are significantly more effective than Gd-DTPA as relaxation agents and possess advantageous magnetic behaviour [25-27]. Notably, iron oxide NPs also exhibit increased blood half-life, biodegradability, and non-toxic nature. Iron oxide NPs are indeed commonly used as T2 or negative CAs in MRI, making images darker. These T2 contrast agents negatively affect the pathological site due to their large magnetization values and magnetic behaviour [28]. Despite the contrasting ability and biocompatibility, iron oxide NPs based CAs are limited due to certain artefacts that may arise due to calcium deposits, internal bleeding, the presence of other metals, and signal voids present in the area being examined [29,30]. Due to these reasons, CAs such as Feridex® and Endorem® were discontinued, and positive contrast agents are much preferred in clinical settings, knowing their limitations [31].
Thus, there is an urgent need to develop non-toxic CAs, and in this context, several approaches have been adopted. Studies have shown that iron oxide NPs can be used to produce positive contrast in MRI by modifying the surface and the size [29,32]. Iron oxide NPs have been stabilized with functional groups to maintain stability and improve solubility. The surface coating can change the surface chemistry and electronic environment around Fe ions by affecting nearby water protons to relax through electronic exchange and influencing the local magnetic field fluctuations to enhance T1 relaxation. Biocompatible materials were mostly chosen as coating materials, depending on the type of application intended [33]. Typically, there are two primary methods for coating magnetic iron oxide NPs: in situ coatings, where the NPs are coated during the synthesis process, and post-synthesis coatings, applied after the NPs have been produced [34,35]. Carbon or silica shells provide a non-magnetic, chemically stable protective layer around MNPs, significantly enhancing their utility in various applications. Carbon has been demonstrated to offer greater stability against acids, bases, and oxidation compared to traditional polymer or silica shells [36]. Carbon-coated magnetic materials present a promising strategy for achieving enhanced chemical and mechanical stability, regardless of fluctuations in pH or temperature in the cellular microenvironment. This carbon shell with a magnetic core facilitates dipole-dipole interactions with water protons, enhancing T1 relaxation rather than T2 [37,38]. Moreover, the outer carbon shell of these MNPs can be readily modified with a variety of drugs and ligands, facilitating effective drug delivery and targeting of cancer cells [39]. While studies have investigated the impact of coating thickness on the relaxometry properties, the effect of the surface functional groups on the physicochemical properties of the iron oxide NPs remains largely unexplored [40]. The primary drawback of coating molecules on iron oxide NPs is the increase in particle size, often exceeding 100 nm following the coating process.
In this study, we present a simple one-pot hydrothermal synthesis procedure for the synthesis of variable degrees of carbon dot-coated iron oxide NPs. The relaxometry properties can be altered mainly by adjusting the composition of the precursors. Citric acid was chosen as the stabilizing agent for the synthesis of iron oxide NPs and also as a precursor for the formation of carbon dots [41]. The varying citric acid concentration will help to synthesize smaller-sized particles. Carbon coating can enhance the absorption intensities of MNPs in the visible light spectrum by creating oxygen defects on their surfaces [42,43]. Moreover, it reduces uniaxial anisotropy on the particle surface and lowers the average blocking temperature [44,45]. However, achieving stability and dispersion of the particles is often challenging due to their inadequate surface physicochemical properties. Carbon dots have several advantages, such as chemical inertness, high photostability, good solubility, tuneable surface, and photoluminescent properties. These features have made carbon dots a promising candidate for nanomedicine applications [46-48]. In this study, we hypothesized that the surface of iron oxide NPs can be modified with carbon dots to generate positive contrast. The effect of the surface modification on the physicochemical properties was also studied by varying the number of carboxylate molecules. The designed CDs@Fe2O3 can act as potential T1 and T2 CAs as well as an optical imaging fluorophore, which will enable the tracking of the path of the contrast agent in vivo. Thus, the fabricated NPs will serve as a biocompatible label-free multi-mode contrast agent.
Materials and methods
Iron(II) chloride tetrahydrate (FeCl2.4H2O) and Iron (III) chloride hexahydrate (FeCl3.6H2O) were obtained from Sigma-Aldrich, India. Urea (CH4N2O), Citric acid (C6H8O7), XTT sodium salt (C22H16N7NaO13S2), and DMEM medium were purchased from Himedia Laboratories, India. All chemicals used were of analytical grade (≥99 %) and were utilized in their original form. Double-distilled water was employed for all experiments.
Synthesis of carbon dot-coated iron oxide nanoparticles
One pot hydrothermal synthesis method was utilized to fabricate carbon dot-coated iron oxide nanoparticles (CDs@Fe2O3). The precursors used for the reaction are 1:2 molar ratios of 0.1M iron II and 0.2 M iron III chloride, 1 g urea, and various concentrations of citric acid added in 50 mL of double-distilled water. CDs@Fe2O3 were synthesized at five different concentrations, with varying concentrations of citric acid precursor from 1, 2, 3, 4 and 6 grams. The concentration of iron (II & III) chloride and urea did not change. The resultant samples were marked as C1, C2, C3, C4, and C5, respectively. The precursors were mixed and transferred to a Teflon-lined stainless-steel autoclave, which was maintained at 150 °C for 12 h, allowing the precursors to undergo a polymerization reaction. The reaction mixture was left to reach room temperature, and the polymer solution was purified by dialyzing against distilled water for 6 h in a dialysis membrane with 14 kDa MWCO (molecular weight cut-off). The resultant dispersant was then heated in a hot air oven to remove moisture at 150 °C for 10 h. The samples were then powdered and stored/used for further studies.
Sample characterization
The crystalline nature of the CDs@Fe2O3 was investigated using a Smartlab Rigaku X-ray diffractometer, Japan. A Bruker-Alpha FTIR spectrometer (USA) was used to analyse the nature of chemical bonding. The X-ray photoelectron spectra (XPS) were recorded with VG Multilab 2000-Thermo Scientific K-alpha, USA. The magnetic nature of the samples was analysed using Lakeshore, USA VSM (vibrating sample magnetometer). Spectrometric characterization was done with a Shimadzu (Japan) UV-1800 spectrophotometer for absorption spectra, and a Jasco (FP-3800, Japan) Fluorimeter was used for fluorescent spectra. Surface morphology and size analysis of the NPs was carried out by Scanning electron microscope (SEM), JEOL JSM - 7600F, Japan. Particle size distribution and zeta potential analysis were performed using the Malvern, UK Nano-Zs particle size analyser. To perform T1 and T2 imaging, a GE, USA, Signa HDxT 1.5 T MRI scanner was used. Optical imaging was done using the Perkin-Elmer (USA) IVIS-Lumina LT in vivo imaging system. Surface area, pore size, and pore volume were studied using BETSORP Max BET Surface area analyser, Microtrac BEL, Japan.
Haemolysis assay
The haemolysis assay was carried out following the established protocol from the literature, with slight modifications [36]. Fresh mice blood was collected in EDTA tubes, and red blood cells (RBCs) were collected by discarding the supernatant after centrifugation at 3000 rpm for 10 min. The RBCs were then washed five times with phosphate buffer saline (PBS). The purified RBCs were then diluted with PBS buffer 10 times. From the diluted RBCs, 100 μL was added into tubes containing 900 μL water (+ve control), 900 μL PBS (-ve control) and 9 μL PBS with sample concentrations ranging from 0.05 to 0.4 mg. Both control and samples were incubated at 37 °C for 2 h. After incubation, tubes were centrifuged at 12000 rpm for 2 min, and the absorbance (A) of the supernatants was measured at 541 nm to calculate the haemolysis, %, usingEquation (1):
Phantom magnetic resonance and fluorescence imaging
Phantom MRI was performed in a 96-well plate, where all five samples of CDs@Fe2O3 were added in varied concentrations of 10, 20, 30, 40, 60, 80 and 100 μg/mL. Following the standard protocols, both T1 and T2 weighted MR imaging were performed [49]. For T1 weighted imaging, the FLAIR sequence was opted to get a slice thickness of 2 mm with the parameters TE (echo time) = 14 ms, TR (repetition time) = 3000, FOV (field of view) = 24 24, and variable T1 was obtained from 400 to 2000 ms. T2 weighted imaging was performed using a turbo spin-echo sequence, acquiring 2 mm thick slices with a TE range of 10-100 ms, TR of 10000 ms, a field of view of 24 24 cm, and an echo train length of 12. The optical imaging ability of the CDs@Fe2O3 was validated in vitro using the IVIS small animal imaging system, PerkinElmer. In vitro imaging for sample C3 was carried out in a 96-well microtiter plate with increasing concentrations of 0, 15, 30, 45, 60, 75 and 90 μg/mL. The fluorescent images were acquired using a GFP filter at 460 nm.
Cell viability assay
XTT assay was performed to analyse the cytotoxic effects of the synthesized CDs@Fe2O3 against FL (Follicular lymphoma) cells. In brief, 2×106 cells were seeded per well in a 96-well plate and incubated at 37 °C, 5 % CO2 overnight. The cells were treated with 50, 100, 200, 300 and 400 mg/mL concentrations of CDs@Fe2O3 (Sample C3) for 24 h. After the incubation, the XTT reagent was added in dark conditions and then incubated for 5 h at 37 °C. DMSO was added and mixed well to dissolve the formazan crystals. The absorbance of the sample was measured with a microplate reader at 450 nm. The assay was done in triplicate.
Hematology and histopathology
The animals were divided into 3 groups, each consisting of 6 mice. Group I was kept as control, while Group II and Group III were injected with the synthesized CDs@Fe2O3 (Sample C3). The animals in groups II and III were administered 500 μL of C3 through the tail vein, and the blood samples and organs were collected 1 h post-injection for group II and 24 h post-injection for group III. After a specified time, animals were anesthetized, the blood samples were collected by the retro-orbital method in EDTA-stabilized tubes, and the hematology assays were performed following the standard protocols [22]. A complete blood count, which includes measurements of red blood cells (RBC) and white blood cells (WBC), was performed using a modified Neubauer hemacytometer. The microhematocrit technique was utilized to determine the percentage of packed cell volume (PCV), and the concentration of haemoglobin was measured using the acid hematin method. Blood urea nitrogen (BUN) levels were obtained from serum samples analysed with semi-automatic analysers to evaluate the nephrological functions.
For histopathological analysis, body parts, including the heart, kidney, liver, and spleen, were harvested, and small pieces from them were taken and fixed in Bouin's solution and transferred to 10 vol.% formalin-neutral buffer [22]. Standard histological procedures have been utilized for tissue processing. After embedding with paraffin blocks, the organs were transversely sectioned to the desired thickness of 3 to 5 μm by using a rotatory microtome, and the sections were positioned on glass slides. The obtained sections were stained with H&E (Hematoxylin and eosin) for routine histopathological procedures and viewed under a light microscope at 4, 10, and 100a magnifications to investigate histoarchitecture.
Ethical statement
All the animal experiments were conducted following the ethical guidelines of the Institutional Animal Ethics Committee (IAEC) of Chettinad Hospital and Research Institute (CHRI), Chettinad Academy of Research and Education (CARE). The reference number was IAEC3/Proposal: 27/A.Lr: 09. The IAEC approved all the experimental protocols related to the study.
Results and discussion
Physicochemical characterizations
The crystalline structure and phase identification of the synthesized samples were investigated by powder X-ray diffraction technique (Figure 1A) [50]. The purity of the samples was confirmed by the strong and sharp Bragg's reflections assigned to (104), (113), (116), (018) and (208) planes of the Miller indices, respectively (PDF # 33-0664). The diffraction peaks can be indexed to pure rhombohedral α-Fe2O3 NPs [51]. Further, the peak cantered at 23.06° is attributed to the highly disordered carbon atoms, thereby confirming the presence of the amorphous carbon shell over the iron oxide core. The XRD peaks correspond well with the standard XRD pattern for α-Fe2O3 (hematite) (JCPDS card no 87-1166), confirming that synthesized iron oxide NPs are hematite in nature. No phase change was observed among the samples, and all the samples showed the same peak positions, as shown inFigure 1A. By utilizing the Scherrer equation, it was possible to determine the crystallite size of CDs@Fe2O3. The calculated crystallite sizes of the samples are represented inTable 1. It was observed that the variation in the amount of citric acid has a considerable impact on the size of the NPs. Citric acid added to the reaction mixture not only acts as a precursor for the formation of carbon dots but also acts as a reducing agent in the synthesis of iron oxide NPs. The C3 sample shows a smaller crystalline size compared to other samples.
The functional groups and chemical bonding nature of the samples were analysed using FTIR, as shown inFigure 1B. The broad absorption band at 3327 cm-1 corresponds to the O-H stretching vibration of carboxylic acid [52]. The carboxylic functional groups impart a hydrophilic nature and enhance the stability of the CDs@Fe2O3. The asymmetric and symmetric sharp bands at 1638 cm-1 and 1041 cm-1 correspond to the N-H bending and C-N stretching of amine. The peaks observed at 1160, 1437 and 1635 cm-1 correspond to C-C, C-O and C=C bond vibrations of carbon. The absorption band at 534 cm-1 indicates the presence of the Fe-O bond of the iron oxide NPs. All of the obtained FTIR bands revealed the formation of carbon dots and iron oxide NPs, and no notable differences were seen in the absorption bands of the samples from C1-C5 (Figure 1B) [51].
A dynamic light scattering technique was applied to determine the hydrodynamic diameter (dH) and zeta potential of the synthesized CDs@Fe2O3, and the results are exhibited inTable 1. The recorded hydrodynamic diameters of the samples C1-C5 are represented inFigure 1C. The increasing concentration of citric acid typically leads to the formation of smaller carbon dots due to its increased nucleation rates. Lower (1 and 2 g) citric acid concentration makes larger particles due to incomplete reaction with urea and poor stabilization. 3 g citric acid shows the smallest NPs, and good surface functionalization is due to sufficient carbon formation and uniform coating. The higher concentrations of 4 and 6 g of citric acid produce particles of larger sizes due to their inappropriate carbon masking, making a thicker shell. From the results, C3 is certainly the optimal value of citric acid to be added to synthesize the lowest possible dH of the CDs@Fe2O3. Changes in concentrations (higher or lower) will result in the synthesis of particles with higher dH. Furthermore, zeta potential results also support the idea that sample C3 has the highest particle stability in comparison to the other samples (Table 1).
With the brief discussions about the effect of the surface chemistry on the size and stability of the CDs@Fe2O3, we speculated that the good affinity of the carboxylate molecules to adsorb on the iron oxide surface would also ideally alter the saturation magnetization (Ms) values of the samples. To support this, we investigated the Ms values of the CDs@Fe2O3 at room temperature by employing vibrating sample magnetometry (VSM). The plotted VSM spectra of the samples are presented inFigure 1D. The obtained Ms values are less than those of the commercially available iron oxide NPs. The Ms value of the sample C3 is slightly higher than the other samples (Table 1). This variation in the Ms values can be attributed to the canted spins in the oxidized layer of the iron oxide NPs. Magnetic particles in the nanoscale tend to act as a core-shell material, with the core composed of aligned spins and the latter composed of disordered spins. The layer with the disordered spins is also known as the dead layer or oxidized layer. These disordered spins can interact ferromagnetically with the external magnetic field and can contribute to the total magnetization value. As evident from the stability values of the Zeta potential, C3 has plenty of carboxylate molecules, which also means that the surface spin canting will be higher in C3 than in the other particles. The Ms values of the other samples are lower than C3, as they are larger and do not have a high amount of carboxylate molecules on their surface, as C3.
Surface chemistry and morphology
XPS was employed to ascertain the chemical composition, oxidation states, and bonding of the C3. The full range XPS spectrum presented inFigure 2A represents the photoelectron peaks of Fe 2p, C 1s, O 1s, and N 1s at 709.96, 284.04, 531.01 and 399.75 eV, respectively.
The high-resolution spectra of each element are presented inFigure 2B-E. The binding energies at 723.72 and 709.96 eV correspond to Fe 2p1/2 and Fe 2p3/2. In the O 1s spectrum, the photoelectron line at 529.99 eV binding energy corresponds to the Fe-O bonding, and the binding energy at 531.01 and 532.00 eV is attributed to C=O and C-O. From the Fe 2p and O 1s, it can be deduced that the valence state of the iron oxide core is +3 and -2, respectively. This affirms that the iron oxide is in the hematite phase, and there is no sign of impurity or phase transformation [51]. The high-resolution spectra of the C 1s spectra exhibit four peaks at 284.04 eV (C=C), 283.01 eV (C-C), 285.07 eV (C-O), 287.85 eV (C=O). The peaks at 284.04 and 283.01 eV binding energies correspond to the C sp2 and C sp3 peaks of graphitized and amorphous carbon. The N 1s spectra have two peaks at 399.75 and 401.03 eV, which represent the C-N-C and the amine groups (N-H) present at the surface of the carbon dots and also support the formation of CDs@Fe2O3 [51]. The obtained XPS results are in line with the results obtained from XRD and FTIR studies. The morphology of the C3, as observed in the SEM, is shown inFigure 2F. The SEM image showed that particles of uniform spherical structure are homogeneously distributed. The mean diameter of the C3 was calculated as 55 ± 4.5 nm.
Surface area analysis
The synthesized CDs@Fe2O3 (C1-C5) surface area was investigated using the Brunauer-Emmett-Teller method (BET) (Figure 3A). The mean pore diameter, pore volume, and surface area of the samples are summarized inTable 2. To understand the porous characteristic of the samples, nitrogen gas adsorption-desorption isotherms were obtained for all the samples, and the plots were identified as type IVa isotherms having an H1 hysteresis loop according to the IUPAC standard [53]. The type IVa isotherm suggests the presence of inhomogeneous mesopores in samples, which is additionally verified by the Barrett, Joyner, and Halenda (BJH) pore size distribution plots (Figure 3B). The BJH method confirmed the existence of mesopores, revealing that the pore diameters of the samples are between 2 and 50 nm. FromTable 2, it can be observed that the samples are mesoporous, with different pore diameters and volumes, because of the variation in the concentration of citric acid. As already outlined in DLS and XRD, the samples are of different sizes; hence, the surface area characteristics have also differed, confirming the initial observations.
Spectrophotometric and fluorometric analysis
The synthesized CDs@Fe2O3 showed a broad absorption band ranging from 430 to 465 nm centered at 452 nm, depicting the formation of the carbon dots around the core of the MNPs as represented inFigure 4A. A similar absorption peak was observed for all the samples (C1-C5). The obtained absorption range is solely attributed to the presence of carbon dots because neither citric acid nor urea absorbs within the wavelength range [54,55].
The broad absorption peak was further analysed by steady-state fluorescence measurement to find out the emission maxima, followed by excitation spectra to get the exact excitation wavelength of C1 to C5. Interestingly, the emission maxima (λemmax) were 550 nm for C1 and showed a continuous reduction of 547 nm for C2, 464 nm for C3, 454 nm for C4 and 440 nm for C5 (Figures 4B to 4F). A similar shift was observed when excitation spectra were recorded fixing λemmax at their (C1-C5) individual maxima. A narrow and sharp excitation peak was observed at 512 nm for C1, 510 nm for C2, 392 nm for C3, 375 nm for C4, and 358 nm for C5 (Figure 4B to 4F). Increasing the concentration of citric acid, keeping the concentration of Fe(II), Fe(III), and urea fixed in the synthesis, can modify the surface chemistry of the fluorescent particles, enhance surface passivation or altering the types of functional groups presented on their surface. This alteration can impact the electronic environment, resulting in a shift in the fluorescent wavelength. Urea functions as the source of carbon, along with citric acid and amino functional groups that contribute to the fluorescence properties. It was reported that urea and citric acid are involved in an open and closed reaction system during the hydrothermal process to produce a polymer-like structure that occupies the surface of the magnetic cores [49]. This peak shift confirms the binding mechanism of carbon dots on the surface of MNPs [49]. The fluorescence imaging capability of C3 was further analysed by the IVIS optical imaging system.
Phantom magnetic resonance imaging
A phantom MRI study was executed to evaluate the contrasting ability of the CDs@Fe2O3 (Figure 5). The samples were prepared in different concentrations and then imaged under the MRI scanner following the standard protocols. The obtained images were analysed with the DICOM image processing program supplied by the manufacturer, demonstrating that the samples exhibited good positive contrast in T1 weighted MRI and negative contrast in the T2 weighted images. The carbon coating acts as a barrier between Fe2O3 NPs, preventing agglomeration. Dispersed particles, with minimal clustering, exhibit reduced magnetic inhomogeneity, allowing them to contribute to T1 (longitudinal) relaxation more effectively. The image intensities significantly rise in T1 weighted images for increasing concentration and moderately decrease in T2 weighted images for limited concentration (Figure 5A to 5D). The C3 sample shows better T1 relaxivity than other samples and the brightening effect started to increase sharply after 30 μg ml-1 concentration (Figure 5A to 5B). The T2 intensity is almost similar for C2-C4 (Figure 5C-D) and slightly higher than the other two samples, C1 and C5. Therefore, the synthesized CDs@Fe2O3 show a prominent T1 effect rather than T2. Evaluating the contrasting ability of CDs@Fe2O3 requires determining the longitudinal (r1 = 1/T1) and transverse (r2 = 1/T2) relaxivity values. These values were derived from T1 FLAIR images with varying TI ranging from 400 to 2000ms and T2 TSE images with TE ranging from 10 to 100 ms. Graphs were plotted to show the T1 and T2 intensities in relation to the increasing concentrations of CDs@Fe2O3, assessing both molar relaxivities (Figure 5B). The r1 molar relaxivities for C1, C2, C3, C4 and C5 were calculated as 4.1±0.8, 5.9±0.4, 6.2±1.0, 5.1±0.5 and 3.8±0.7 mM-1 s-1, respectively. The r2 values were calculated for C1, C2, C3, C4, and C5 as 29.3±1.9, 31.2±1.1, 32.1±1.1, 32.8±1.6 and 28.9±1.0 mM-1 s-1, respectively. The calculated data show better relaxivities compared to commercially available contrast agent Gd-DOTA (r1 and r2 relaxivities of 2.94 and 3.60 mM−1 s−1, respecttively), as reported by Thirumalai et al. [47]. The r2/r1 ratio for sample C1-C5 lies in the range of 10 > r2/r1 > 3.
The relaxivity results show that CDs@Fe2O3 has enhanced T1 relaxation rather than T2, as observed earlier in the intensity. This is due to carbon coating influencing the relaxation of nearby water protons through the electronic exchange, affecting the local magnetic field fluctuations, favouring T2 relaxation by facilitating dipole-dipole interactions with water protons. This indicates the potential of the CDs@Fe2O3 to act as an efficient twin-mode T1 and T2 CAs in MR imaging, and sample C3 shows higher T1 signal intensity than the other samples. The C3 features an optimal CD coating that is thin enough to maintain water access to the iron core, hydrophilic enough to attract water, and stable enough to prevent aggregation. This ideal combination leads to improved T1 relaxivity. This observation follows those obtained from the zeta potential analysis, hydrodynamic diameter, XRD patterns and VSM. Hence, sample C3 was used for further experiments.
Phantom optical imaging
The fluorescence ability of the C3 was assessed by phantom optical imaging. Sample C3 was taken in increasing concentrations (0, 15, 30, 45, 60, 75 and 90 μM ml-1) and imaged in a small animal imaging system, as shown inFigure 6. It was observed that the fluorescence intensity gradually increased with the higher concentration of C3 (Figure 6A-C). The images obtained from optical imaging were adjusted to reflect photons per second per square centimetre per steradian (p s-1 cm-2 sr-1) following the elimination of background signals. A gradual increase in intensity was noted with rising concentrations of C3 of CDs@Fe2O3.Figure 6D illustrates the graphical depiction of average radiant efficiency alongside the concentrations of CDs@Fe2O3, where the slope indicates the average radiant efficiency of 6.549 × 106 p/sec/cm2/sr per mM. The data clearly showed that the CDs@Fe2O3 (sample C3) can be used for multimodal imaging.
In vitro and in vivo toxicity assessment
In vitro, cytotoxicity assessment of synthesized CDs@Fe2O3 (Sample C3) was evaluated by XTT cell viability assay performed on FL cell lines. The obtained data shows that the synthesized particles show similar cell viability compared to the control (Figure 7A). Sample C3 does not exhibit significant toxicity, even at the highest concentration. As a result, it was determined that the toxic effect of ferrite nanoparticles was significantly mitigated by the carbon coating, indicating that the synthesized CDs@Fe2O3 (Sample C3) are biocompatible. A haemolysis assay is essential to investigate the hemocompatibility of the prepared NPs and ensure in vivo safety. For the haemolysis assay, the CDs@Fe2O3 (Sample C3) was taken in five different concentrations ranging from 0.05, 0.1, 0.2, 0.3 and 0.4 mg/ml. Based on the absorbance values, the percentage of haemolysis was calculated, and it was found to be <2 % for the sample with a high concentration of the CDs@Fe2O3. This value shows that the synthesized particle is haemocompatible and complies with the ASTM E2524-08 standard parameter (Standard test method for analysis of hemolytic products of NPs). As per the ASTM standards, NPs that show less than 5 % heamolytic activity are considered haemocompatible. The absorbance spectrum of the positive control and negative control, along with the photograph of the haemolysis activity, is represented inFigure 7B. The haemolysis percentage obtained for the CDs@Fe2O3 (C3) aligns well with the standard value, emphasizing the remarkable hemocompatibility of the CDs@Fe2O3.
Haematological analysis is an essential method for evaluating the toxic effects of nanomaterials by studying their interactions with blood and its compounds. The hematologic parameters, such as packed cell volume (PCV), RBC, WBC, and haemoglobin were investigated from the whole blood. The nephrotoxicity of the administered NPs was assessed by monitoring the BUN values in serum samples collected from animal models divided into 3 groups. The obtained results are shown inTable 3. A moderate decrease in RBC count after 1 h of injection, which increased to normal after 24 h of injection, was noted, and the PCV was also increased for 1 h of injection. The decrease in RBC level is due to the higher oxygen level and decreased haemoglobin metabolism regulated by erythropoietin secretion in the kidney. The leukocyte count was recorded in all three groups and showed an increase over time, indicating an enhanced immune response to the injected foreign material. However, despite the increase in WBC count, it is deemed insignificant as the levels remained low and within the normal range. This suggests that the majority of the injected CDs@Fe2O3 are able to evade the immune response.
Kidneys play a major role in renal filtration, and the results show that the BUN values increase after 1 h and return to normal after 24 h. Thus, the BUN evaluation shows that the metabolism of Fe2O3NPs in the kidneys depends on time. Even though there were slight variations in the hematologic values, all the values were in the normal range.
Histopathological examination of the mice was conducted to analyse the impact of the CDs@Fe2O3 (C3) on different organs, utilizing Haematoxylin and Eosin (H&E) staining, as illustrated inFigure 8. No significant changes were noted between the control group and the other two groups administered with CDs@Fe2O3. From the photomicrographs of the liver and kidney, it can be seen that the architecture of the liver and kidney in the treatment groups is similar to that of the reference group. In the liver, the hepatocytes exhibit clear cytoplasm, while in the kidney, the papilla, inner medulla, and cortex appear normal. No abnormal lesions were found in the heart tissues, and no changes were observed in the red pulp or white pulp of the spleen. Thus, it can be inferred that the administration of the C3 sample of CDs@Fe2O3 doesn't result in any significant changes in the mice organs investigated and thus is safe to be administered in vivo.
Conclusion
In summary, this study has developed a promising method for synthesizing carbon dot-coated iron oxide nanoparticles (CDs@Fe2O3) and standardizing the concentration of citric acid that regulates both the reaction process and the size of the iron oxide nanocluster. It was identified that changes in the surface chemistry of iron oxide NPs can have considerable effects on the physicochemical properties. The characteristic properties of the synthesized CDs@Fe2O3 highlight the stability attained through the specific concentration of citric acid employed in this synthesis method. Based on the findings from phantom imaging, it is clear that the CDs@Fe2O3 exhibit superior magnetic and fluorescent properties, making them suitable for use as an effective dual-mode MR imaging agent in conjunction with fluorescent imaging. The concentration of precursors to obtain maximum contrast enhancement has been optimized, and the same has been confirmed with the findings from characterization studies such as FTIR, XRD, VSM, zeta potential analysis, and DLS measurements. The synthesized particles were found to be negligibly toxic and possess good hemocompatibility. The present study offers insights into the biocompatibility of the CDs@Fe2O3 (C3) synthesized via a robust method. This was assessed by evaluating haematological parameters and kidney function through BUN levels and conducting histopathological analyses of various organs in mice. Thus, the synthesized CDs@Fe2O3 shows promising results for being considered as an alternative biocompatible multimodal contrast agent.