The samples were subjected to Powder X-ray Diffraction using a PANalytical X-Pert Pro X-ray diffractometer having Cu as anode material and having Kα1 as 1.54060 Å. Figure 2a depicts the XRD plot retrieved for [email protected]2 NPs and Fig. 2b shows the XRD pattern obtained for bare TiO2 NPs. Figure 2b showcases anatase TiO2 NPs obtained on calcination at 400 °C. The peak at 25.3764° confirms the  plane of the anatase phase. The entire pattern matched well with the JCPDS pattern: 01-073-1764 confirming the peaks of anatase TiO2 NPs. Different planes recognized from the plot are  at 25.322°,  at 36.998°,  at 37.863°,  at 38.600°,  at 48.064°,  at 53.975°,  at 55.094°,  at 62.756°,  at 68.870°,  at 70.330°,  at 75.141° and  at 82.756°. Fig. 2a demonstrates the XRD peaks of [email protected]2 NPs and here the iron-capping has been done on the same TiO2 NPs whose XRD obtained is shown in Fig. 2b. We clearly observe that both the patterns are identical and thus [email protected]2 NPs also possess the similar anatase phase, although the peaks are a little broadened. Whether the XRD pattern of the core–shell NPs will match with that of the core NPs, this depends upon the thickness of the shell. The possibility of [email protected]2 NPs having the similar XRD pattern can be attributed to the thin layer of coating on the TiO2 NPs. The incident x-rays can easily penetrate the shell thickness of ~10 nm and below and thus almost a similar pattern is obtained. Moreover, a careful investigation indicates that one extra peak has emerged at 35.631° which corresponds to plane  of cubic Fe2O3 as denoted by JCPDS pattern: 00-039-1346. This is justified as the coating of ferrous fumarate (Fe2+) was done onto the TiO2 NPs. The crystallite size calculated using Debye–Scherrer’s equation stated the size of ~12 nm for TiO2 NPs and ~17 nm for [email protected]2 NPs. Furthermore, Fig. 2c depicts the superimposed XRD patterns obtained in both Fig. 2a, b. One can easily tell that the entire XRD plot of [email protected]2 NPs has been shifted up in intensity. This is a striking observation that the core–shell [email protected]2 NPs retain the similar XRD pattern of the core TiO2 NPs. The basic idea of core–shell formulation is to have a uniform coating while still conserving the properties of the core. The layer formed must never be too thick; otherwise, it will overshadow the features of the core25.
The High-Resolution Transmission Electron Microscopy (HR-TEM) images of TiO2 NPs are shown in Fig. 3a, b whereas Fig. 3c, d demonstrate the HR-TEM images of [email protected]2 NPs. Figure 3a shows that the average size of the TiO2 NPs appear to be ~10 nm. This goes along with the crystallite size calculated from the respective XRD plot. The particles are nearly spherical and there is a uniform size distribution. The interplanar distance lies between 0.3 and 0.4 nm, more precisely 0.37 nm as observed in Fig. 3b. The inset shows the magnified view of the region selected to measure the interplanar distance. In Fig. 3c, we can clearly see the core–shell formulation of the NPs. Now, the NPs are no longer isolated, but agglomerated and an external layer/capping is distinctly visible. The darker portions indicate the presence of element with lower atomic number and here relate to the core i.e. TiO2 NPs. The lighter portions indicate the presence of an element with a higher atomic number which is Fe2+ form of the iron in this case and corresponds to the shell. The inset displays the magnified view of the image which has been used to measure the thickness of the layer. The scale of the inset is 50 nm and with this, it is deduced that the layer is at most 10 nm thick. In Fig. 3d, two different interatomic spacings of 0.37 nm and 0.29 nm can be seen overlapping in most of the regions. Figure 3e depicts the Selected Area Electron Diffraction (SAED) pattern for TiO2 NPs which matches well with the corresponding XRD pattern. Fully bright dots and distinct rings are clearly visible. This confirms the highly crystalline nature of TiO2 NPs. However, the SAED pattern for [email protected]2 NPs in Fig. 3f consists of diffuse rings which signifies their less crystalline nature. Once again, this can be confirmed from the respective XRD pattern in Fig. 2a. Thus, the findings obtained from XRD, HR-TEM and SAED analysis are in good agreement with each other.
The Energy Dispersive X-Ray Analysis (referred to as EDX or EDS or sometimes EDAX) confirms the presence of elements Oxygen (O), Titanium (Ti) and Iron (Fe) in [email protected]2 NPs as shown in Fig. 4a. Similarly, Fig. 4b depicts the presence of Oxygen (O) and Titanium (Ti) in TiO2 NPs. Table 1 outlines the weight percentage and atomic percentage of all the elements in both the samples. The Vibrating Sample Magnetometer (VSM) studies of both [email protected]2 NPs and TiO2 NPs are shown in Fig. 5. The inset of Fig. 5 illustrates the pure diamagnetic nature of anatase TiO2 NPs. The room temperature magnetism induced for the applied magnetic field is a linear graph with a negative slope indicating diamagnetism for TiO2 NPs. The plot of the diamagnetic behavior is similar to the previously reported result26. However, the VSM plot of TiO2 NPs capped with an iron supplement capsule shows superparamagnetic behavior. Beketova et al.27 decorated TiO2 nanotubes with Fe3O4 NPs which also reported superparamagnetic behavior at room temperature. This is a first in itself effort to magnetically modify the NPs using commercially available iron supplements manufactured for human consumption. Thus, [email protected]2 NPs can be driven to the target sites with the help of external magnetic fields. Table 2 summarizes the hysteresis parameters of [email protected]2 NPs obtained via VSM against Magnetization (emu) vs Applied Magnetic Field (Oe).
For the drug release studies, we have investigated the release behavior under different pH levels. The following terminology has been used to indicate the samples in different pH solutions as shown in Table 3.
For both the acidic and the normal mediums, the study was undertaken for three days. The choice of dialysis tube also affects the pattern of drug release. Figure 6 represents the drug release profile of T4 and F4 at pH 4.4 with cumulative release percentage plotted along the y-axis and the time (in minutes) along the x-axis. The pH around carcinogenic tumors is usually acidic, that’s why most of the chemo drugs are designed to deliver faster around acidic environments. Moreover, the orally administered drugs reach first into the stomach after consumption, where the highly acidic medium breaks it down into its components from where the molecules enter into the bloodstream. In our case, the initial release in the first two hours was much faster as compared to the total study of 3 days. T4 and F4 released upto 29.2% and 25.58% respectively at the end of 120 min. T4 showed the cumulative release of 60% in 14 h and finally reached the maximum release of 93% at the end of our study. F4 displayed a rather controlled release pattern presenting approx. 50% cumulative release after 26 h. The maximum release % recorded for F4 at the end of three days is 83%. The inset in Fig. 6 provides the drug release pattern for the first 300 min. Thus, even under the acidic medium, both T4 and F4 showed a sustained release pattern and this attribute can be utilized for the controlled delivery of the drug. Likewise, Fig. 7 demonstrates the drug release profile of T7 and F7 at pH 7.4. The pH of normal blood is generally considered to be between the range 7.35–7.45. The NPs to be used as drug carriers are normally expected to exhibit controlled but faster drug release under acidic environments and less to no release under normal pH. By this we can assure lesser drug wastage during the circulation and more effective drug release at the target locations. Under normal pH, we can see that in the span of three days, the maximum cumulative release percentage was 25% and 18% for T7 and F7 respectively. No further release was observed and F7 has outperformed T7 in the context of least drug release in the normal pH. The inset in Fig. 7 shows the initial release pattern in the first 300 min. T7 presented 5.87% release and F7 showed 9.34% cumulative release in the initial 300 min. Furthermore, the samples were also tested under the basic medium of pH 9 and as expected, an overall release of 9.4% was observed for T9 and 8.9% was observed for F9 as shown in Fig. 8. This study was only stretched for 300 min as no appreciable release was seen after 120 min. This study confirms that the drug release rate of both TiO2 NPs and [email protected]2 NPs is pH-dependent. The maximum release has been detected in the acidic medium and the least release in the basic medium. Thus, both types of NPs are suitable candidates to be used as drug-delivery agents. [email protected]2 NPs exhibited a more controlled drug-release behavior and therefore, the maximal release attained by them is less than TiO2 NPs under all the pH levels.
In-vitro study conducted by Kadivar et al.28 suggests that 99.56% of the Imatinib is released within 20 min in 0.1 N HCl solution. In comparison to this observation, both the NPs synthesized by us demonstrate much controlled and sustained release of the drug (Imatinib) which is very beneficial for an effective chemotherapeutic treatment. If the drug is retained for a longer time, this would minimize the drug dosages required which would further control the side-effects. The results of the in-vitro study revealed the attainment of maximum drug release under an acidic environment by these NPs, suggesting that they undergo relatively faster decomposition thereby promoting faster release at low pH level than at neutral pH level. CaCO3 NPs demonstrate similar pH-responsive behaviour and have been extensively studied for drug delivery applications. They exhibit low toxicity, slow biodegradability and when functionalized, they not only augment the effectiveness of drug-delivery, but also enhance drug-loading, tumor-targeting and thermodynamic stability29. Thus, NPs need to be improved for showcasing multiple benefits.
Figure 9 showcases the UV–visible plots for both the NPs taken at 0 h i.e. at the beginning of the study and at 48 h (at the end of the study), respectively. ‘T’ represents the TiO2 NPs and ‘F’ represents the [email protected]2 NPs. The inset of Fig. 9 shows the comparison of the absorbance of the NBT solution recorded at 254 nm over three different intervals of time i.e. 0 h, 24 h and 48 h respectively in the presence of both the NPs separately. It is observable that there was no significant decrease in the absorbance of the characteristic peak at 254 nm over the period of time. The plots of TiO2 NPs-NBT solution and [email protected]2 NPs-NBT solution taken over different instants of time almost overlapped. Moreover, no colour change was detected by observing the samples with naked eye. Thus, the ROS detection test conducted by us for both the NPs validate that no ROS is generated by these NPs under human physiological conditions which confirms their biosafety. Basante-Romo et al.30 modified TiO2 NPs with functionalized multiwalled carbon nanotubes and evaluated them for their level of toxicity. The toxicity studies were performed on female albino rats and no adverse effects were observed after the 10-days study. No mortality was produced. All the clinical signs were normal suggesting that no changes occurred in the cells or tissues exhibiting normal architecture of the organs. Thus, the modification of TiO2 NPs diminishes the chances of them being destructive. The coating of these NPs with a biocompatible material and other sorts of surface modifications assure that these TiO2 NPs won’t be displaying their ROS generation ability sans irradiation. Since these NPs are not irradiated when used for drug-delivery applications, the chances of them resulting in lethal ROS are very low.
The usage of NPs as drug-delivery agents belong to new-era medical techniques. A lot of research is going on to ensure the suitability of these NPs for the said purpose. This very idea is still in its infancy and it has to successfully pass a plenty of pre-clinical and clinical trials to be actually able for a certified medical usage. Talking about ensuring targeted drug delivery of chemotherapeutics, emphasis should be laid on a variety of factors including the mitochondrial response, the oxidative stress, the receptors present, any sort of inherent or induced drug resistant response of the cellular components and many more. Furthermore, any kind of toxicity offered by the NPs should also be dealt with great care. The toxicity of the NPs does not lie only in the formation of ROS, but the size and shape of the NPs are also the factors of grave concern. The NPs should be optimally synthesized to have optimal size and shape which offer least accumulation in the tissues and organs. Different techniques to functionalize the NPs, to modify their surface properties, to optimize their size and shape, to ensure adequate amount of drug loading onto them, to improve their antioxidant characteristics and to make them biocompatible, should be devised. Thus, adequate steps should be taken to ensure the biosafety of the NPs to be used as drug-carriers and to further improve their drug-delivery response. Once satisfactory results are achieved from in-vitro studies, only then more systematic approaches can be explored to guide in-vivo research and better correlate the properties of nanoparticles with their biological effects.