Synthesis, characterization and In-vitro studies of CNT/Gd 2 O 3 hybrid structure

Carbon Trends 11 (2023) 100272 Contents lists available at ScienceDirect Carbon Trends journal homepage: www.elsevier.com/locate/cartre Synthesis, characterization and In-vitro studies of CNT/Gd 2 O 3 hybrid structure Rima Paul a , b , ∗ , Dipanwita Chatterjee c , Lopamudra Das Ghosh a , d , Venkatesha Narayanswamy a , Mahander Pratap Singh a , Manish Agarwal e , Deepshikha Ghosh f , Mithun Radhakrishna f , Chandra Sekhar Tiwary g , h , Ivo Provazník b , Kamanio Chattopadhyay a a Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India b Department of Biomedical Engineering, Brno University of Technology, Brno 61600, Czech Republic c Materials Research Centre, Indian Institute of Science, Bangalore 560012, India d Department of Biomedical Engineering, Texas A& M University, Texas 77843, USA e Computer Services Centre, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India f Department of Chemical Engineering, Indian Institute of Technology Gandhinagar, Gujarat 382355, India g Metallurgical and Materials Engineering, Indian Institute of Technology Kharagpur, West Bengal 721302, India h Department of Material Science and Nano Engineering, Rice University, Houston, Texas 77005, USA a r t i c l e i n f o Keywords: Carbon nanotube Gadolinium oxide Paramagnetism Biomedical application a b s t r a c t Carbon nanotubes have been explored in various fields of science and technology due to their unique properties. However, their toxic nature put limitations to its consideration for the bio-medical applications. In our work, we report the synthesis of paramagnetic CNT/Gd 2 O 3 hybrid nanostructures through easily scalable electrochemical deposition technique. The nanostructure has shown significant longitudinal relaxivity of 18.93 mM − 1 s − 1 for water protons indicating its possibility to be used as an MRI contrast agent. The in-vitro cytotoxicity test on the normal (HaCaT) and cancerous (HeLa) cells reveal that CNT/Gd 2 O 3 hybrid nanostructure is bio-compatible in comparison to the pure CNTs, which is further supported by our theoretical studies using MD simulations. 1. Introduction Since their discovery in 1991, carbon nanotubes (CNTs) [1] , have become one of the prime studied material owing to their unique properties [2–4] . CNTs find numerous usages in energy, environment, electronics, composites and sensor applications [ 5 , 6 ]. The range of application of CNTs further enhanced by surface modification through functionalization with a variety of molecules which tunes the physical and chemical properties. Such engineering of the CNT surfaces can make it more effective for applications in the field of optoelectronics [ 6 , 7 ] light-emitting diodes [8] , photocatalysis [9] , photovoltaic devices [10] , to design sensor systems and to fabricate electrochromic devices [11] . Surface modification of the CNT walls can increase its effectiveness and also make it a promising candidate for various biomedical applications [12–14] . But, in-spite of all fascinating properties, carbon nanotubes are known to cause toxicity in certain living cells [15–18] which limits its extensive application in the field of medical science. Although, the interaction between CNTs and biological environment can be engineered with the help of morphology, purity and functionalization [ 19 , 20 ], the CNTs are found to exhibit different levels of toxicity depending on their synthesis ∗ Corresponding author. E-mail address: [email protected] (R. Paul) . route, shape, aspect ratio, concentration, composition, functional group attached, level of oxidation and dosage [ 21 , 22 ]. Their hydrophobic nature also makes CNTs less biocompatible [23] and is known to cause necrotic and apoptotic cell death. Researchers have reported toxicity of multi walled carbon nanotubes (MWCNTs) and single walled carbon nanotube (SWCNTs) arising due to their length and diameter. The incorporation of metal impurities during their synthesis is also reported to induce toxicity to the cells [24] . However, decorating nanoparticles, quantum dots or functional moieties to their surfaces may reduce the toxicity of the CNTs, making them suitable for cellular life [19] . As reported by earlier researchers, surface modification of CNTs make them eligible to be used in the cellular imaging for the diagnosis and biomarker and targeted drug delivery for cancer therapies or tumour xenografts [25– 28] . Hence, coating CNT with biocompatible metal oxide can solve the problem of cytotoxicity considerably and can find potential use in nano biotechnology and biomedical applications. Gadolinium oxide nanoparticles on the other hand have proved to be the potential candidate for targeted drug delivery in cancer treatment. Gadolinium compounds are now replacing boron-10 in neutron capture therapy (NCT) [29] . Gd 2 O 3, is attracting interest of the researchers behttps://doi.org/10.1016/j.cartre.2023.100272 Received 2 January 2023; Received in revised form 21 April 2023; Accepted 23 May 2023 2667-0569/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ) R. Paul, D. Chatterjee, L. Das Ghosh et al. Carbon Trends 11 (2023) 100272 cause of its possibility to be used for multicolour marking, time resolution of the luminescence and high spatial resolution of MRI which could enhance the efficiency of contemporary biomarkers [30] . The Gd 2 O 3 nanoparticles have been used in magnetic resonance imaging (MRI) as they exhibit either super-paramagnetism or paramagnetism and involve T1 as well as T2 relaxation thereby offering themselves to be used as multimodal contrast agents for in-vivo imaging [ 31 , 32 ]. However, the advantage of using CNTs over other nanoparticles owes to their unique structures and their sp 2 hybridization, which enable them to get easily functionalized with the respective ligand or therapeutic moiety. The first SWCNT based contrast agent called “gadonanotube ”has been reported by Sitharaman et al. [33] followed by other in the field of bioimaging [ 34 , 35 ]. The SWCNTs are functionalized in various ways and the modified structures have shown excellent properties to be considered as potential MRI contrast agents. But these SWCNT based hybrid structures possesses several challenges and imperfections to be rectified before implementation in the medical field. Modified MWCNT stood good in this respect and has drawn the attention of the researchers to be considered a better contrast agent [36] . As mentioned by Vittorio et al. [37] , the compatibility of CNTs depends on their purity, size and functionalization. CNTs are known for their toxicity and Gd-based materials already have biomedical applications. But hybrid structures of carbon nano-allotrope and Gadolinium have been reported to have induced 10–90-fold greater relaxavity with a lower dose and enhanced MR imaging than the conventional Gd-based contrast agents [ 18 , 38 ]. As, mentioned earlier, the first CNT based contrast agent called “gadonanotube ” was reported by Sitharaman et al. [33] where ultra-short single walled carbon nanotubes encased superparamagnetic Gd 3 + n -ion clusters within their framework. Such gadonanotubes demonstrated high relaxivities and potential for targeted magnetic field-induced hyperthermia. Surface modified carbon nanotubes have the reported ability to translocate the cell membrane and for MRI technology such translocation effect might be critically important for cell trafficking applications involving cellular tagging [39] . Such CNT-Gd constructs have been demonstrated as potential cell probe for T1 weighted imaging. Consequently, a hybrid structure of CNT/ Gd 2 O 3 has drawn our attention to explore its potential to be used as a positive MRI contrast agent. To our knowledge all CNT-Gd 2 O 3 hybrid material reported so far involved functionalization of CNT surfaces with ligands or acidic functional groups but in this work, we deposited Gd 2 O 3 directly to their surfaces through electro chemical deposition technique. We have characterized the synthesized material to know their composition and morphology and performed in-vitro cytotoxicity assay on normal (HaCaT) and cancerous cell line (HeLa) with respect to the pure CNTs and the CNT/Gd 2 O 3 hybrid nanostructure. As per our studies depositing paramagnetic Gd 2 O 3 on CNT walls reduces their toxicity and the hybrid structure exhibited higher longitudinal (r1) and the transverse (r2) relaxivity compared to many of the reported values of the Gd-based contrast agents. 2. Materials and methods 2.1. Materials Forests of multi walled carbon nanotubes were synthesized on silicon wafers using water assisted chemical vapour deposition (WACVD) technique. Details of the preparation of such CNTs can be found elsewhere [40] . These CNTs were decorated with Gd 2 O 3 nanoparticles following electrochemical deposition route. The electrochemical deposition was carried out using Metrohm AUTOLAB potentiostat consisting of a threeelectrode system. CNT forest on a silicon wafer was used as the working electrode (cathode), a platinum foil as counter electrode and Ag/AgCl as a reference electrode. Gd 2 O 3 was electrodeposited onto the surface of CNTs at room temperature, from an aqueous solution of gadolinium nitrate hexahydrate (Gd (NO 3 ) 3 ·6H 2 O, Alfa Aesar, purity 99.9%) and boric acid (H 3 BO 3 , Merck, Assay 99.5%). Addition of boric acid was made to adjust the pH of the solution to 2.24. The electrodeposition process was carried out at a potential of − 0.85 V. This electrodeposition potential was considered based on the linear sweep voltammetry studies over the potential range of 0 to − 1.00 V at a scan rate of 20 mV/min [41] . The synthesized CNT/Gd 2 O 3 nanostructures were then washed repeatedly with de-ionized water using an ultrasonictor bath (GT Sonic, 40 kHz) to remove excess of Gd 2 O 3 present if any, filtered and allowed to dry at room temperature. 2.2. Instruments and characterizations The morphological analyses were done using the scanning electron microscopy (ESEM Quanta 200, FEI and SEM, ZEISS ultra 55), transmission electron microscopy (FEI Tecnai T20 S-TWIN microscope, operated at 200 kV acceleration voltage) and high angle annular dark field imaging in scanning transmission electron microscopy mode (HAADF-STEM imaging) using FEI Titan G2 60–300 microscope, operated at an acceleration voltage of 300 kV and spot size 6. The X-ray diffraction (XRD) pattern was obtained from the pure CNT and the as-synthesized CNT/ Gd 2 O 3 hybrid nanomaterial using X-ray diffractometer (X-Pert PRO, PANalytical) employing a Cu K 𝛼radiation. The compositional analyses were carried out using electron dispersive x-ray analysis (attached with ESEM), Fourier transform infrared spectroscopy (Nicolet 6700 from ThermoFisher Scientific) and Raman spectroscopy (Renishaw Raman spectroscope with 633 nm wavelength laser excitation). The magnetic property of the hybrid nanomaterial was obtained using a Lakeshore vibrating sample magnetometer (VSM). To quantitatively validate the hybrid material as a potential MRI contrast agent, we have measured the relaxation of water protons in the presence of the hybrid material using an NMR spectrometer. For the NMR studies, the as synthesized hybrid material was dispersed in 95% D 2 O and 5% H 2 O. All the relaxation measurements of water protons in the presence of CNT/Gd 2 O 3 hybrid structures was conducted at a temperature of 298 K using AV400 Bruker NMR Spectrometer operated at a resonance frequency of 400 MHz. The longitudinal or the spin-lattice relaxation ( T 1 ) and the transverse or spinspin relaxation ( T 2 ) times were measured using the inversion recovery and Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence respectively. To avoid radiation damping, only one scan was used to acquire the data. The concentration of Gd in the CNT/Gd 2 O 3 water dispersion used in the NMR experiments was determined by inductively coupled plasma optical emission spectrometer using Thermo Fisher Scientific iCAP 7200 ICP-OES at a commercial laboratory (Ramiah Advanced Testing Laboratory, Bangalore). A standard stock of 100 ppm was prepared using Gd (NO 3 ) 3 ·6H 2 O salt in 5% HNO 3 solution. Then, 1.1 mg of the synthesized CNT/Gd 2 O 3 hybrid nanomaterial was added to 100 mL of the 5% HNO 3 solution to find out the amount of Gd present per litre of the dispersion. 2.3. Cell culture and in-vitro cytotoxicity assay To determine the biocompatibility of the CNT/Gd 2 O 3 hybrid nanostructure, in-vitro cytotoxicity analysis was performed. HaCaT and HeLa cell lines obtained from the ATCC and used for cytotoxicity evaluation studies were cultured in DMEM F12 and DMEM media respectively with 10% FBS(v/v), 1% penicillin-streptomycin and 2 mM glutamine at 37 °C with 5% CO 2 . All the cell culture media, FBS, glutamine and antibiotics were procured from Gibco, USA. The toxicity of both pure CNT and CNT/Gd 2 O 3 hybrid at different concentration of 6.0, 12.5, 25.0, 50.0 and 100.0 μg mL − 1 were tested against both cell lines for 24 h and 48 h. In this study pure CNT and CNT/Gd 2 O 3 hybrid study groups are indicated as (P) and (M) respectively. The cells grown in the absence of CNT and CNT/Gd 2 O 3 hybrid served as the control for the study. The cells were trypsinised using 0.025% trypsin EDTA (Gibco, USA) and 5 ×10 3 cells were seeded in each wells of 48-well plate. MTT solution diluted in serum free culture medium was added to the cells at a concentration of 1 mg mL − 1 and incubated for 3 h. To dissolve the formazan crystals formed, MTT solution was replaced by DMSO and the absorbence was measured at 570 nm to calculate the cell viability, using microplate 2 R. Paul, D. Chatterjee, L. Das Ghosh et al. Carbon Trends 11 (2023) 100272 reader (Synergy HT, BioTek instrument). The study was conducted in triplicates. 2.4. Fluorescence microscopy Based on the cell viability data from MTT assay, 25 𝜇g/ml was selected as the particle concentration and all the cell studies were performed for this dose. The cells were grown in the presence of pure CNT and CNT/Gd 2 O 3 hybrid structure for 24 h and 48 h. The qualitative viability was performed at 24 h and 48 h respectively using live dead assay kit (Life Technologies, USA) following manufacturer’s protocol. The images were captured at an effective magnification of 100X using Fluorescence microscope (Olympus Z1). The cells fluorescing green were live while the dead ones were represented by red fluorescence. 2.5. Scanning electron microscopy The ultra-structural characterization of cells’ morphology was performed after 24 h and 48 h of culturing cells in the presence of pure CNT and CNT/Gd 2 O 3 hybrid nanostructure using a scanning electron microscope (SEM, ZEISS ultra 55), after sputtering with gold ( ∼15 nm thick) at 5.0 kV accelerating voltage. 2.6. Molecular dynamics simulations The Molecular dynamics simulations were carried out using CHARMM27 force field in GPU accelerated NAMD v2.11 with a time step of 1fs. Hexagonal carbon sheets and phosphatidylcholine (POPC) membranes were created using "Nanotube Builder" and "Membrane Builder" extensions of VMD v1.9.2 respectively [42] . All simulation images were rendered in VMD v1.9.2. A 100 Åx 100 ÅPOPC membrane was created and solvated in TIP3P water. The system was energy minimized to ease any initial steric strain and subsequently simulated in the NPT ensemble for 1e6 steps at 300 K. Temperature and potential energy were quickly stabilized in less than 100 ps. Volume stabilization was done in less than 500 ps. It is worth mentioning that extending the simulations did not alter stability. Graphene sheets of size 100 Åx 100 Åwere created and merged with the energy minimized solvated POPC membrane structure ensuring that any overlapping water molecules were removed. The system was initially equilibrated for 1e6 steps at 300 K, and then extended for 10e6 more steps. The volume was stabilized only after 5e6 steps, and remained stable for the rest of the 5e6 steps. Langevin thermostat and barostat were used in all simulations with damping coefficients of 1ps − 1 and 100ps − 1 respectively. 3. Results and discussions MWCNT has already proven its ability to be used as a platform for designing MRI cell probe [39] . Several researches have been done earlier on the chemical functionalization of MWCNTs to enhance their dispersibility by adhering carboxyl and hydroxyl group to their surfaces [ 43 , 44 ]. But in this work, we have decorated the walls of highly aligned MWCNTs with Gd 2 O 3 nanoparticles in a simple way without any prior chemical functionalization. Gd 2 O 3 was electrodeposited onto the surface of pure CNTs at room temperature and thereafter HaCaT and HeLa cells were treated separately with pure CNTs and CNT/Gd 2 O 3 hybrid material to study their cytotoxicity. To observe the surface modification of CNTs after electrodeposition of Gd 2 O 3 , electron microscopy analyses were conducted. MWCNTs having length of several microns can be seen in SEM micrographs of the MWCNTs in its pure form in Fig. 1 a and after the electrodeposition of Gd 2 O 3 onto their surfaces in Fig. 1 b respectively. The EDS analysis as shown in Fig. 1 c, carried out over a randomly selected area, highlighted in red of the hybrid nanostructures, clearly indicates its elemental composition. The bright field TEM micrograph of the pure CNTs and that of the CNT/Gd 2 O 3 nanostructures has been shown in Fig. 1 d and Fig. 1 e respectively. The diameter of these pure MWCNTs varies between 10 and 20 nm. In the Fig. 1 f, HAADF-STEM micrograph of the hybrid structures shows distinctly the bright spots corresponding to the Gd atoms throughout the length of the CNTs. Similar spots due to Gd were reported by earlier researchers who prepared a gadolinium functionalised CNTs through a different synthesis route [39] . The formation of CNT/Gd 2 O 3 hybrid nanostructure can be further interpreted from the SAED pattern ( Fig. 1 g), wherein the diffraction from (100) graphitic plane of carbon i.e. CNTs (ICSD#031,170) and (31 3), (11 2), (401), (20 1) planes of monoclinic phase of Gd 2 O 3 (ICSD#160,226) are clearly revealed. The presence of these phases can also be observed in the XRD pattern of the synthesized CNT/Gd 2 O 3 hybrid material as shown in Fig. 2 a. The broad peak around 25.8° is due to the overlapping of (002) & (110) planes of CNT and Gd 2 O 3 respectively. The pattern shows the reflections due to (401), (11 2) and (311) of Gd 2 O 3 along with another overlapping peak due to CNT (100) and Gd 2 O 3 (31 3). These observations from the XRD data well supports the inference drawn from the SAED pattern discussed earlier. In Fig. 2 b the FTIR spectra of the pure CNT and the hybrid nanostructure have been presented. An absorption ∼1400 cm − 1 of the raw MWCNTs corresponds to the C –C stretch, originated from the graphitic CNT structure [45] . For the CNT/Gd 2 O 3 hybrid nanostructure, the stretching vibration of O –H bonding found ∼3442 cm − 1 is due to the moisture absorbed by KBr while preparing the sample for FTIR analysis. The stretch around 2350 cm − 1 corresponds to the background CO 2 in the spectrometer. The appearance of a small absorption ∼1660 cm − 1 is related to the stretching vibrations of C = C of the CNTs [46] . The formation of gadolinium oxide is confirmed from the stretch around 547 cm − 1 , which is attributed to the Gd-O bonding [ 47 , 48 ]. The magnified version of the stretch attributed to the Gd-O bonding of CNT/Gd 2 O 3 hybrid nanostructures has been presented in the inset of Fig. 2 b for clarity. Raman spectrum provides information about the structure of CNTs and it has been shown in Fig. 2 c. The D-band ( ∼1334 cm − 1 ) arises due to the presence of defects and sp 3 hybridized carbon atom. The quality of the nanotube which means the presence of disorders is inversely related to the height of this band. The G-band ( ∼1575 cm − 1 ) of the spectra quantifies graphitization and is related to the sp 2 hybridized carbon atom of the CNT wall. I D /I G indicates the ratio of intensities of the disorder induced D-band to the symmetry allowed G-band of the graphitic material and it measures the degree of disorder in the sp 2 hybridized carbon structure. The I D /I G for the pure CNT is 1.11 and the ratio has reduced to 1.05 after decorating CNT surfaces with Gd 2 O 3 . This decrease in I D /I G indicates that the degree of disorder in the CNTs have decreased, which in turn means greater degree of graphitic crystallinity has been obtained [49] . The decrease in I D /I G is also an indication that no covalently bonded functional group has been introduced to the CNT surfaces [50] while synthesizing the hybrid structure. The change in the degree of disorder may be attributed to slight defect supression of lattice [51] after decorating CNT walls with Gd 2 O 3 . There is also a blue shift of the D peak by 3 cm − 1 for the hybrid nanostructures, indicating a charge transfer between CNTs and Gd 2 O 3 [52] . To utilize a material as an MRI contrast agent, it is necessary to understand its magnetic properties. The dependence of the magnetic moment of both pure CNTs and CNT/Gd 2 O 3 hybrid nanostructures on the applied magnetic field obtained at a room temperature is shown in Fig. 2 d. The pure CNT shows diamagnetic nature with respect to the applied magnetic field (H) varied in the range − 2T < H < 2T, whereas CNT/Gd 2 O 3 hybrid exhibits paramagnetic nature. The M-H curve of the synthesized hybrid structure at 300 K revealed negligible magnetic remanence and coercivity. Till an applied field of 2T, no magnetic saturation is observed which indicates that the hybrid material doesn’t possess any ferromagnetic phases. The net magnetization (M) value for the hybrid structure at 2T is 6.1 emu/g, which is quite appreciable and owes to the high-spin magnetic moment of the 4f-electrons of Gd 3 + [32] . The 3 R. Paul, D. Chatterjee, L. Das Ghosh et al. Carbon Trends 11 (2023) 100272 Fig. 1. (a, b) SEM micrographs of pure CNTs and CNT/Gd 2 O 3 hybrid material respectively (c) EDS spectra showing the constituent elements present in a randomly selected area of the hybrid material (d, e) bright field TEM micrographs of pure CNTs and CNT/Gd 2 O 3 hybrid material respectively (f) dark field TEM micrograph clearly shows the deposition of Gd 2 O 3 nanoparticles on to the CNT surfaces (g) SAED pattern from the corresponding TEM micrograph indicates the formation of Gd 2 O 3 on the CNT surfaces. Fig. 2. (a) XRD pattern of the CNT/Gd 2 O 3 hybrid material showing reflections from the planes of CNT (C) along with that of Gd 2 O 3 (G) (b) FTIR spectra of pure CNT and CNT/Gd 2 O 3 hybrid nanostructure; inset shows stretch due to Gd-O bonding, with magnification (c) Raman spectra of pure CNTs and CNT/Gd 2 O 3 hybrid structure (d) dependence of magnetization on the applied magnetic field indicating the conversion from diamagnetic to paramagnetic nature of CNTs upon depositing Gd 2 O 3 nanoparticles on to their surfaces. 4 R. Paul, D. Chatterjee, L. Das Ghosh et al. Carbon Trends 11 (2023) 100272 Fig. 3. 1/T vs. Concentration of Gd plot. The dotted lines are the linear fits to the data points and the relaxivity values of water protons were determined from the slope of these fitted lines. value of magnetization has been calculated considering the mass percentage of Gd in the hybrid material. The amount of Gd present in the hybrid nanostructure is determined using the inductively coupled plasma optical emission spectrometry (ICP-OES) analysis. There are significant reports [ 53 , 54 ] on the use of CNT based magnetic nanocomposites for various biomedical applications viz. hyperthermia therapeutic strategies, targeted drug delivery and as MRI contrast agent. Researchers also reported magnetic materials viz. polyaspartic acid coated Gd 2 O 3 nanoparticles, graphene oxide-Fe 3 O 4 nanoparticle composite, graphene oxide-CoFe 2 O 4 nanoparticle composite, poly (methyl vinyl ether-altmaleic acid) coated Gd 2 O 3 nanoparticles, etc. for biomedical use and those materials showed a magnetization values comparable to what we achieved with CNT/Gd 2 O 3 hybrid nanostructures [ 32 , 55-57 ]. Therefore, a clear possibility exists for the hybrid structure of CNT/Gd 2 O 3 to be used in biomedical application, especially as an MRI contrast agent. As mentioned earlier, the advantage of using CNTs over other nanoparticles for biomedical applications lies in the fact that they have unique structures owing to their aromatic nature that helps them to get easily functionalized for improved biocompatibility and drug delivery applications. Fig. 3 shows the Gd-concentration dependant spin-lattice relaxation (T1) and spin-spin relaxation (T2) values of water protons, measured using 400 MHz NMR spectrometer. The concentration values are the different dilutions with respect to the concentration of Gd. The longitudinal (r 1 ) and the transverse (r 2 ) relaxivity has been calculated to be 18.93 mM − 1 s − 1 and 51.26 mM − 1 s − 1 respectively. These relaxivity values have been estimated from the slopes of the linear fit in the concentration vs. inverse of relaxation time (1/T) plots. These values of r 1, r 2 and r 2 /r 1 are very much close to the reported value of paramagnetic gadolinium oxide contrast agents, prepared by G.H. Lee and his team [32] and are higher than those of commercially available Gd-chelates [ 58 , 59 ]. Several Gd 2 O 3 based contrast agents have earlier been reported [ 57 , 60-62 ] which have r 2 /r 1 values above 2. The ratio r 2 /r 1 defines the potential of a material to be used in providing a positive or a negative contrast. A high value of r 2 /r 1 = 2.71 reported here may significantly reduce the contrast effect because for a highly sensitive T 1 MRI contrast agent the value should be close to unity [61] . The hybrid nanostructure provided in this study has shown significant enhancement in the relaxivity values of water protons. The determined longitudinal (r1) and the transverse (r2) relaxivity being 18.93 mM − 1 s − 1 and 51.26 mM − 1 s − 1 respectively are higher compared to the some of the reported values of the Gd-based materials. Caravan et al. [59] reported various Gd(III)-complex based contrast agents whose longitudinal relaxivity varies between 2.09–10 Fig. 4. Dose dependant temporal cytotoxicity evaluation of normal and cancerous cells on exposure to pure CNT (P) and CNT/Gd 2 O 3 hybrid (M) nanostructure. (a-b) represent the viability percentage of HaCaT and HeLa respectively on exposure to different concentrations of nanostructures for 24 h (left Y-axis) and 48 h (right Y-axis). Data are represented as means ± SEM (standard error of the mean) for three independent biological replicates ( n = 3). Statistical significance was performed using Two-way ANOVA, followed by post-hoc Tukey test; ∗ ∗ p < .01, ∗ ∗ ∗ p < .001, ∗ ∗ ∗ ∗ p < .0001 respectively. mM − 1 s − 1 and transverse relaxivity between 2.34–15.9 mM − 1 s − 1 . Such relaxaivities were obtained under different temperature, pH and 1 H frequencies. Paramagnetic ultrasmall Gd 2 O 3 NPs prepared by Park et.al. [60] showed 9.9 mM − 1 s − 1 and 10.5 mM − 1 s − 1 as r1 and r2 value respectively. Fortin et al. [61] reported ultra-small Gd 2 O 3 nanoparticles capped with polyethylene glycol and diethylene glycol that showed r1, r2 to be 9.4 mM − 1 s − 1 ,13.4 mM − 1 s − 1 and 6.4 mM − 1 s − 1 ,15.2 mM − 1 s − 1 respectively. The core-shell hybrid structure of Gd 2 O 3 -polysiloxane reported by Bridot et al. [62] exhibited longitudinal relaxivity of 4.4 mM − 1 s − 1 and transverse relaxivity value of 28.9 mM − 1 s − 1 . The commonly used positive contrast agents, Gd-DOTA shows lesser relaxivities in comparison. Difference in the experimental conditions is likely to influence the relaxivity values, however, a qualitative analysis shows the efficiency of our synthesized CNT/Gd 2 O 3 hybrid nanomaterial when compared with the reported literature values as reflected from Table 1 . The cytotoxic response of cells on exposure to different concentrations of pure CNT and CNT/Gd 2 O 3 hybrid has been assessed in-vitro on HaCaT ( Fig. 4 a) and HeLa ( Fig. 4 b) after 24 h and 48 h. The in-vivo cytotoxicity evaluation on human and rat has reported significant elimination of Gd from the body system within first 24 h [ 63 , 64 ]. Hence, the cytotoxicity evaluation for our study was performed in-vitro for 24 h and 48 h. The toxicity shown by pure CNT is higher for both HaCaT and HeLa cells with respect to dose and time. Analysis of MTT assay results indicate that the particle concentration of 25 μg mL − 1 is safe to be administered for MRI imaging without any significant cytotoxicity for CNT/Gd 2 O 3 hybrid. The quantitative cell viability analysis is completely in corroboration with the fluorescence imaging of the qualitative live-dead cell assay. Images of live-dead cell assay have been furnished for 25 μg mL − 1 of pure CNT (P) and CNT/Gd 2 O 3 (M) in Fig. 5 . As evident from the fluorescence images, the exposure of both HaCaT ( Fig. 5 a) and HeLa ( Fig. 5 b) cells to CNT/Gd 2 O 3 resulted in significantly reduced cytotoxicity compared to pure CNTs after 24 h and 48 h. The scanning 5 R. Paul, D. Chatterjee, L. Das Ghosh et al. Carbon Trends 11 (2023) 100272 Table 1 Comparison of relaxivities of different Gd-based contrast agents. Materials r 1 (mM − 1 s − 1 ) r 2 (mM − 1 s − 1 ) r 2 /r 1 Ref. CNT/Gd 2 O 3 hybrid 18.93 51.26 2.71 Present article B-21,326/7 6.78 7.77 1.14 [59] BOPTA 4.39 5.56 1.27 [59] DOTA 3.56 4.75 1.33 [59] DTPA-BBA 4.08 6.01 1.47 [59] 16-DTPA-PN –OH 3.5 4.4 1.26 [59] DO3A-L4 5.19 7.29 1.04 [59] DOTPMe 2.09 2.34 1.12 [59] PC2A 7.5 8.3 1.11 [59] EDMP 11.1 15.9 1.43 [59] Phos-L1 10.0 14.3 1.43 [59] DTPMP 8.6 11.8 1.37 [59] Paramagnetic ultrasmall Gd 2 O 3 NPs 9.9 10.5 1.06 [60] PEG-Gd 2 O 3 9.4 13.4 1.42 [61] DEG-Gd 2 O 3 6.4 15.2 2.40 [61] PEG-SPGO 0.1 7.6 81.6 [61] Gd–DTPA 4.1 4.7 1.1 [61] Gd-DOTA 4.1 4.9 1.20 [62] Hybrid Gd 2 O 3 NPs 4.4 28.9 6.80 [62] Fig. 5. Fluorescence imaging showing the effect of CNT/Gd 2 O 3 hybrid and pure CNTs on the (a) HaCaT and (b) HeLa cells; Red indicates dead cells and green resembles live cells. Pure CNT and CNT/Gd 2 O 3 hybrid study groups are indicated as (P) and (M) respectively; concentration of the materials being 25 μg mL − 1 . electron micrograph of the HaCaT cells has indicated a shrinkage in the cell size ( Fig. 6 a), but there is no evidence of cell damage or rupture in the presence of CNT/Gd 2 O 3, unlike that observed in the HeLa cell line ( Fig. 6 b). The pure CNTs, on the other hand has shown an adverse effect on both cell lines as expected and it eventually worsen with time. To summarize, the toxicity effect of the synthesized CNT/Gd 2 O 3 nanostructure has been found to be acute in the case of cancerous cells compared to the normal healthy cells and this can be accounted to the surface modification of the CNTs as reported earlier [65] . Furthermore, the toxicity shown by the CNT/Gd 2 O 3 hybrid nanostructures over pure CNTs on normal cells is significantly less and is in corroboration to the reported literature [66] . The toxicity noted on the exposure of HaCaT to the pure CNT is acute and higher with increase in particle concentration and time. The present study has elucidated that the optimum concentration of 25 𝜇g/ml of CNT/Gd 2 O 3 is safe to be used in the human body system as contrast agent for enhancing the contrast in MR imaging. The toxicity of the pure CNTs and the hybrid structure have also been explained with MD simulation. Fig. 7 shows the snapshots obtained from Molecular Dynamics simulations of POPC layers in water and on the surface of graphene sheets. The outer layer of HaCaT cells are modelled as POPC layers as detailed in the simulation methods section. CNT’s are hydrophobic in nature and upon electrodeposition of Gd 2 O 3, CNT surfaces shows a hydrophilic behaviour. By understanding the interaction between POPC layers and surfaces of different chemistry we can infer about the behaviour of HaCaT cells on these surfaces. The behaviour of HaCaT cells on CNT/Gd 2 O 3 surface is qualitatively akin to its behaviour 6 R. Paul, D. Chatterjee, L. Das Ghosh et al. Carbon Trends 11 (2023) 100272 Fig. 6. SEM micrographs showing the impact of CNT/Gd 2 O 3 hybrid material and pure CNT on the (a) HaCaT and (b) HeLa cells after 24 h and 48 h. Pure CNT and CNT/Gd 2 O 3 hybrid study groups are indicated as (P) and (M) respectively; concentration of the materials being 25 μg mL − 1 . in water (both are of hydrophilic nature) whereas its behaviour on CNT’s is similar to its behaviour on graphene (both are of hydrophobic nature). To reduce the size of simulation, we have chosen to model the surface of multi-walled nanotube as a stack of graphene sheets. Fig. 7 a shows the structure of POPC layer in a hydrophilic environment. We observe the POPC layer is intact by exposing the hydrophilic head and burying their hydrophobic tails. However, upon interaction with hydrophobic graphene sheets, we observe that the POPC structure is distorted and the outer hydrophilic layer of POPC is perturbed as shown in Fig. 7 b. The deformation is also known in literature [67– 69] and it strongly indicates that cell membranes would be adversely affected in the presence of CNTs, leading to the death of the cells. New insights can also be obtained by looking at the order metric for the POPC chains. The order metric is the head to tail distance of a single POPC molecule. Fig. 7 (c, d) shows the order metric in water (hydrophilic) and on graphene (hydrophobic) environments. There is a clear indication that the POPC layers are deformed on the hydrophobic graphene surface. It is evident that, on interaction with graphene, more high order POPC are being transformed into low order POPC indicating deformation of the membrane. Fig. 7 e shows the probability distribution of the order metric and indicates that near the hydrophobic graphene surface, the order metric is shifted to lower order validating the observations from Fig. 7(c, d). It is to be noted that for clarity, water is not shown in Fig. 7(a, b and d). 7 R. Paul, D. Chatterjee, L. Das Ghosh et al. Carbon Trends 11 (2023) 100272 Fig. 7. (a) Snaposhot (top view) of POPC layers in water (hydrophilic environment); the environment is similar to CNT/Gd 2 O 3 . POPC layers are intact without any deformation (b) Top view snapshot of POPC layer near hydrophobic graphene sheets; the environment is similar to CNT surface. The POPC layers is deformed leading to the death of HaCaT cells on these surfaces. Red is the hydrophilic head; cyan is hydrophobic tail. Dark blue are sample pair of POPC molecules (c) in water and (d) on the hydrophobic graphene sheets. POPC layers colour coded according to the order metric. Dark blue represents high order indicative of stable bilayer and Red represents low order indicative of the rupture of bilayer. As we move from hydrophilic environment (c) to a hydrophobic neighbourhood (d), we see a decrease in the dark blue colour and an increase in red colour confirming rupture of bilayer. Green indicates intermediate order which is present in both the cases. (e) Probability distribution of head to tail distance in POPC layers. 4. Conclusions CNT/Gd 2 O 3 hybrid nanostructures were synthesized through electrodeposition of Gd 2 O 3 nanoparticles onto the surfaces of the multiwalled carbon nanotubes. Such deposition led to the conversion of diamagnetic nature of the CNTs into the paramagnetic one. Significant enhancement in the relaxivity values of water protons were achieved with such hybrid nanostructure material. The in-vitro studies revealed reduced cytotoxicity for CNT/Gd 2 O 3 hybrid compared to pure CNTs. The hydrophobic nature of pure CNTs contribute to higher cytotoxicity and hence depositing Gd 2 O 3 nanoparticles on CNT surfaces render hydrophilicity thereby reducing the toxic effects on cells. This work thus indicates that unlike pure CNTs, CNT/Gd 2 O 3 hybrid material can possibly be considered as a potential candidate for biomedical therapies and diagnostics. However, elaborate in-vivo studies should be performed to better understand the mechanism and cell compatibility of the hybrid material. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgements Rima Paul would like to extend her sincere thanks to the UGC, Govt. of India for providing Dr. D. S. Kothari Postdoctoral Fellowship. Lopamudra Das Ghosh acknowledges support from the DST Young Scientist fellowship ( YSS/2015/001448 ). We extend our sincere thanks to Dr. Kaushik Chatterjee, IISc for valuable discussions and providing us with the cell culture facilities. Authors would like to acknowledge AFMM, IISc for the microscopy facilities. We also acknowledge with thanks the Spectroscopy Analytical Test Facility, Society for Innovation & Development (SID), IISc for FTIR data acquisition and NMR Research Centre, IISc for the NMR facility. Manish Agarwal conveys thanks to IIT Delhi HPC facility for the computational resources. References [1] S. Iijima, Helical microtubules of graphitic carbon, Nature 354 (1991) 56–58, doi: 10.1038/354056a0 . [2] S. Banerjee, M.J.C. Khan, S.S. Wong, Rational chemical strategies for carbon nanotube functionalization, Chem. Eur. J. 9 (2003) 1898–1908, doi: 10.1002/chem.200204618 . [3] M.S. Dresselhaus, G. Dresselhaus, P. 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