FericipXT-Coated PEGylated Rutile TiO2 Nanoparticles in Drug Delivery: In-vitro Assessment of Imatinib Release

doi:10.21203/rs.3.rs-3401722/v1

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FericipXT-Coated PEGylated Rutile TiO2 Nanoparticles in Drug Delivery: In-vitro Assessment of Imatinib Release

https://doi.org/10.21203/rs.3.rs-3401722/v1

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Study presents a facile synthesis strategy for magnetic field-responsive PEGylated iron-supplement-coated rutile TiO2 nanoparticles (NPs) for stimuli-responsive drug delivery. Imatinib, an anticancer drug, was successfully loaded onto the NPs, and its in-vitro release was investigated under different pH conditions. XRD analysis confirmed the successful synthesis of PEGylated iron supplement-coated rutile titania NPs. HR-TEM studies revealed increased NP size due to coating, PEGylation, and drug-loading. FTIR spectra confirmed the drug loading onto the NPs, while DLS provided hydrodynamic diameter and polydispersity index, indicating appropriate NP synthesis. The PEGylated NPs exhibited negative Zeta Potential, indicating high stability. In-vitro drug-release studies demonstrated controlled release with maximum efficiency under acidic conditions. Hemolysis assay confirmed the safety and biocompatibility of PEGylated NPs. All drug-loaded nanoformulations followed the Peppas-Sahlin model, suggesting Fickian diffusion and Case II relaxation mechanism of drug release. These NPs have potential for targeted delivery and controlled release of chemotherapeutics, minimizing side effects.

Physical sciences/Physics

Physical sciences/Materials science

Physical sciences/Materials science/Nanoscale materials

Physical sciences/Materials science/Nanoscale materials/Nanoparticles

Biological sciences/Biophysics/Nanoscale biophysics

Drug delivery

Titanium dioxide nanoparticles

Imatinib

PEGylated nanoparticles

Sustained drug release

Challenges in cancer therapy and its solution

Cancers, commonly referred to as malignant tumors, comprise uncontrolled growth of the cells on account of mutation in their genetic material. The adverse effects of cancer and the dire prognosis of patients include high mortality, poor quality of life and expensive treatments [1]. Much technological development and research has been done to combat cancer, including chemotherapy, immunotherapy and radiation therapy. However, these techniques are associated with certain flaws, such as low drug concentrations at the tumor site, non-selectivity to sick cells or tissues and low drug efficacy due to their short half-life. The solution to this menace lies in the usage of targeted drug delivery systems. In such systems, the targeted specificity and selectivity of the drug is greatly enhanced. The main advantage of targeted therapy is that the drug concentration of chemotherapy in the tumor environment can be maximized and toxicity to other non-target organs can be minimized [2]. In addition, if the drug is locally delivered then this can improve drug effectiveness by avoiding the long journey required to reach the target site through the hostile environment when the drug is administered by the systemic route. To better understand tumorigenesis and increase drug availability, several vectors have been explored that can deliver drugs in a controlled manner without causing side effects. To date, the versatile nanomaterials used including polymers, lipids, inorganic molecules, hydrogels and other macromolecular scaffolds have led to the development of systems to deliver chemotherapy drugs directly to tumor sites with improved treatment efficiency [3].

Enhanced permeation effect in tumors

General features of the tumor include leaky vasculature and poor lymphatic drainage. Several approaches have been proposed to target tumors, for example, the permeation-enhancing and retention effects where leaky blood vessels promote small-molecule permeation and poor lymphatic drainage allowing storage and accumulation of chemotherapy drugs in tumor cells [4]. Unlike freely diffusing drugs, a nanosupporter can enter tumor tissue through leaky vasculature due to its enhanced permeability and retention effects. In Enhanced Permeation Effect (EPR), the delivered agent is locally released and rapidly absorbed by the cancer cell to perform its function. Targeted tumor cells that express or overexpress specific receptors or antigens can be targeted using ligands such as antibodies or small peptides that recognize and bind to them [5]. Smaller molecules accumulate in the tumor faster than larger molecules, but larger molecules can be retained in the tumor longer. Therefore, size is a dominant factor in controlling drug accumulation at the tumor site [6].

Nanocarriers for targeted therapy

Active targeting refers to the interaction of ligand-receptors after the nanoparticles (NPs) reach the target site through the systemic circulation. Ligand-receptor interaction is possible only if the two components are in close proximity (0.5 nm). Active targeting of tumors can be achieved by functionalizing NPs with proteins, peptides, nucleic acid aptamers, carbohydrates, and other small molecules [7]. To date, several classes of materials for targeted therapy have been developed, including biodegradable polymers, liposomes, dendrimers, nanoshells, and NPs based on nucleic acids. Biodegradable nanoparticles are widely used in cancer therapy due to their high biocompatibility. For targeted delivery, sustained release and site-specific delivery are key requirements. Another important factor is the stability of the NPs to stay longer in the blood and eventually accumulate in tumours [8]. Drug release from these NPs can be controlled by diffusion or swelling followed by mass diffusion or erosion in a time-dependent manner. The release rate can be adjusted by modifying the polymer, developing new polymers, or synthesizing copolymers. The main advantage of these biodegradable polymers is to maintain drug concentrations within the optimal range for longer periods of time and thus increase drug efficacy and improve patient compliance. Thus, targeted therapy is a combination of targeted administration with continuous drug release, allowing maximum use of the drug delivered to the tumor site.

Titanium dioxide nanoparticles (TiO₂ NPs) are being studied extensively for their drug delivery applications. Their properties can be modified by carefully controlling the synthesis parameters. They can be effectively functionalized using different coatings and surfactants. TiO₂ NPs can be tweaked to respond to different stimuli such as light, sound, pH, temperature, etc. and initiate the drug release [9]. In this study, we have investigated the pH-responsive release profile of the chemotherapeutic drug Imatinib from PEGylated iron supplement-coated rutile TiO₂ NPs. The drug release was inspected over acidic, neutral and basic pH levels. Moreover, an attempt to understand the drug release mechanism from different formulations was also made via different mathematical models available.

2.1 Materials

The materials used were Titanium TetraIsopropoxide (TTIP), ethanol, nitric acid (HNO₃), PEG 4000 solution, Phosphate Buffer Saline (PBS) solution of pH 7.4, Acetate Buffer solution of pH 4.4, Borate buffer solution of pH 9.0 and Deionized (DI) water. These were obtained from Sigma Aldrich Pvt. Ltd, Bangalore, India and were used as such without any further purification. Ferrous Ascorbate with folic acid tablets under brand name FericipXT manufactured by Cipla Ltd. were purchased from pharmaceuticals in Chandigarh. Dialysis tubing and clips were purchased from Himedia. IKA-RCT basic IKAMAG safety control magnetic stirrer with hot-plate by Merck, India was used. pH testing kit by Insif, India was used to determine the final pH of the solution. For calcination, a muffle furnace by Insif, India was used. Conical High Alumina Crucibles of volume 30 ml by Thomas Scientific were purchased for calcination at higher temperatures.

2.2 Methods

2.2.1 Synthesis of PEGylated Iron Supplement-Coated Rutile TiO₂ NPs

Rutile TiO₂ NPs were synthesized following the sol-gel method. The procedure of synthesis was the same as adopted by us in our previous study with the only difference being that the quantity of the nitric acid was chosen as such so as to make the pH of the final solution as 1.0 [10]. Further, the TiO₂ NPs were calcined at 800^oC to obtain the desired rutile phase. To enhance the magnetic behaviour of the rutile TiO₂ NPs, they were coated with FericipXT tablets (iron supplement) with 1:1 core:shell ratio. For coating, the procedure mentioned by Mehr et. al [11] was followed. Briefly, an optimal amount of FericipXT tablets were added to 250 ml of DI water. Next, TiO₂ NPs were added followed by dropwise addition of 1M NaOH solution. Finally, the solution was heated to reflux at 90°C. The resulting solution was centrifuged, washed with DI water and calcined at 800^oC. The characteristic white color of TiO₂ NPs was changed to brown, indicating the formation of Fe²⁺ coating on them. For PEGylation, the synthesized iron supplement-coated rutile TiO₂ NPs were added to 1% concentration of PEG 4000 solution and stirred for one day at 750 rpm. Lower PEG concentration was chosen to avoid the growth of a thicker PEG layer on the NPs. For the final step, PEG-coated NPs were centrifuged at 9000 rpm for 30 min at room temperature, washed with distilled water and dried.

2.2.2 Synthesis of Drug-Loaded PEG/Fe@TiO₂ NPs

To encapsulate the NPs with the drug (Imatinib), the procedure performed by Akram et. al [12] was chosen with minor modifications. A fixed amount of the drug was reconstituted in 40 ml of DI water along with the addition of 500 mg of synthesized NPs. The solution was stirred for 2 days and then stored for one day. It was then centrifuged and filtered. In this way, three samples were collected, one with the drug loaded on the rutile TiO₂ NPs (known as the Drug-TiO₂ NPs), the other with the drug loaded on the FericipXT-coated rutile TiO₂ NPs (called the Drug-Fe@TiO₂ NPs) and the third sample was the drug loaded onto the PEGylated FericipXT-coated rutile TiO₂ NPs (known as Drug-PEG/Fe@TiO₂ NPs). To measure the amount of drug in the yield obtained, the absorbance of the filtrate was measured at 254 nm. The amount of the drug successfully retained on NPs was determined as a percentage of the encapsulation efficiency in Eq. (1). The drug loading percentage reflects the mass ratio of the drug to the nano drug that has been synthesized by conjugating the drug with a fixed amount of NPs. % Drug loading was calculated by finding the drug concentrations in the loaded NPs considered for the study divided by the amount of NPs used, and then multiplying by 100 according to Eq. (2).

Encapsulation Efficiency (%) = (W_{Drug on the NPs}/W_{Total drug used}) x 100 --------------(1)

Drug Loading (%) = (Concentration of the drug in the NPs/Weight of the loaded NPs considered for the study) x 100 ---------------(2)

2.2.3 In-Vitro Drug Release by Dialysis Method

In the present study, in-vitro drug release study of the drug Imatinib was studied over three different pH values viz. 4.4, 7.4 and 9.0. Three types of drug-loaded NPs formulations used for the study were Drug-TiO₂ NPs, Drug-Fe@TiO₂ NPs and Drug-PEG/Fe@TiO₂ NPs. Firstly, the calibration curve of the drug (Imatinib) was plotted in three different pH mediums. For this, a stock solution of Imatinib was prepared and dissolved in three different pH buffer solutions. The calibration curve is a plot with concentration (µg/ml) on the x-axis and absorbance on the y-axis. The curve was mathematically analyzed to obtain slope and intercept. pH-dependent release behavior at three different pH levels was investigated. All the three types of drug-loaded NPs formulations were inserted into dialysis tubes having a Molecular Weight Cut Off (MWCO) of 8 kDa each. In total, 9 sets were prepared. 3 sets of dialysis tubes contained Imatinib-loaded TiO₂ NPs (Drug-TiO₂ NPs), other 3 sets of the dialysis tubes contained Imatinib-loaded Fe@TiO₂ NPs (Drug-Fe@TiO₂ NPs) and the final 3 sets of the dialysis tubes contained Imatinib-loaded PEGylated Fe@TiO₂ NPs (Drug-PEG/Fe@TiO₂ NPs). To mimic a neutral pH environment, 3 dialysis tubes containing 5 ml each of all the three types of drug-NPs formulations were dipped into 500 ml of 0.1 M Phosphate buffer solution (pH 7.4) separately. Similarly, for the in-vitro study under an acidic environment, 500 ml of 0.1 M Acetate buffer solution (pH 4.4) was used and likewise, 500 ml of 0.1 M Borate buffer solution (pH 9.0) was used to achieve basicity. The dialysis tubes were hung with the help of clips in their respective beakers containing solutions to imitate different pH environments. The entire setup was stirred at 37^oC. After a fixed interval of time, 3 ml of the dialysate was withdrawn from each of the beakers for monitoring the absorbance of the Imatinib molecules released into the respective buffer at 254 nm with the help of UV-Visible Spectrophotometer. The withdrawn amount of the dialysate was replaced with an equal quantity of the fresh buffer solution to avoid the sink conditions. In each observation, the value of the absorbance obtained was used to calculate the concentration of the drug released into the media using the following expression:

Concentration of the drug (µg/ml) = (Absorbance - Intercept)/Slope ---------------(3)

where slope and intercept were obtained from the calibration curve. Percentage of the drug released and the cumulative percentage of the drug released was calculated using the following methods:

% drug released = (Concentration of the drug/total amount of the drug used) x 100 ----------(4)

Cumulative % = (Volume of the sample withdrawn (ml)/Bath volume (ml)) x d_(t−1) + d_t ----(5)

where d_t is the percent drug release at time t and d_(t−1) is the percent drug release at time (t-1).

2.2.4 Hemolysis Assay

Fresh anticoagulated human blood treated with citric-acid-dextrose solution was added into seven vials in equal quantity. 1 ml suspension of PEG/Fe@TiO₂ NPs was added into the five vials at the concentration of 10 µg/ml, 50 µg/ml, 100 µg/ml, 300 µg/ml and 500 µg/ml, respectively. 0.9% saline solution was taken as negative control and 1% Triton X-100 solution was used as positive control. The blood samples containing the PEGylated NPs, positive control and negative control, respectively were shaken up gently and incubated for 45 minutes at 37^oC. They were then centrifuged for 3 minutes at the rate of 4000 revolutions per minute. After centrifugation, the blood splits into two components, the red part ‘heme’ at the bottom and a transparent plasma as the top layer. The upper clearer solution was taken out from the samples for the absorbance study at 540 nm. The percentage of hemolysis occurring in the samples was calculated by the formula indicated as follows:

% hemolysis = \(\frac{Absorbanc{e}_{Sample} - Absorbanc{e}_{Negative control}}{Absorbanc{e}_{Positive control} - Absorbanc{e}_{Negative control}}\) -----------(6)

2.3 Characterization

The synthesized samples were characterized for X-ray Diffraction (XRD) and High Resolution Transmission Electron Microscopy (HR-TEM). XRD was carried out using Panalytical X'Pert Pro equipped with x'Celerator solid-state detector and manufactured by Panalytical, Netherlands. The sample was firmly placed onto the glass slide which was carefully laid down onto the sample holder. XRD was performed at the Sophisticated Analytical Instrumentation Facility (SAIF), Panjab University, Chandigarh, India. HR-TEM was carried out using Jeol 2100 Plus, Japan at CIL, Panjab University, Chandigarh, India. The powdered sample was dispersed in ethanol and ultrasonicated for about an hour. A drop of the solution was then carefully placed onto the copper grid. The grid was eventually air-dried under room temperature and was used for imaging after half an hour. Ultraviolet-Visible (UV-Vis) spectroscopy was used to perform the absorbance studies. A double beam UV-VIS-NIR Spectrophotometer - Lambda 750 from Perkin Elmer, USA was used for the said purpose. For this, a fixed amount of NPs (in µg) were dispersed in DI water of specific pH. The baseline correction was done before conducting the study. Afterwards, blanking was performed with the help of a reference sample. Since this work involves studying the behaviour of the drug release under different pH levels so the reference samples as well as the original samples were prepared in the respective pHs accordingly. Dynamic Light Scattering (DLS) verified the particle size distribution of the resulting NPs. Vibrating Sample Magnetometer (VSM) was used to analyze the magnetic behaviour of the bare NPs and the drug-loaded different formulations of the NPs. The samples were placed in the sample holder and all the studies were performed at room temperature.

X-ray Diffraction

Rutile TiO₂ NPs were synthesized and were then treated with FericipXT tablets to get a coating over them. Finally, these iron tablets-coated rutile titania NPs were PEGylated to get another layer onto them. Figure 1 demonstrates the XRD plot of Imatinib (drug), FericipXT tablet (iron supplement used for coating rutile TiO₂ NPs), FericipXT-coated rutile TiO₂ NPs in the 1:1 ratio, PEGylated 1:1 FericipXT-coated rutile TiO₂ NPs, Imatinib-loaded TiO₂ NPs, Imatinib-loaded FericipXT-coated rutile TiO₂ NPs and Imatinib-loaded PEGylated FericipXT-coated rutile TiO₂ NPs. Table 1 demonstrates the symbols for different samples which have been used from this section onwards in this paper, wherever necessary. The XRD pattern of T belongs to the pure rutile phase (JCPDS Pattern No. 01-073-1782). The highly crystalline nature of T was lost after the coating with FericipXT tablet. Table 2 highlights the different phases of iron-based compounds appearing onto the surface of TiO₂ NPs after their coating with the iron tablet in 1:1 core:shell ratio. The details of the different phases obtained and their quantification have been obtained after the analysis of the XRD data through HighScore Plus software. The XRD pattern of F is different from that of T and FericipXT because of the formation of iron oxides during the core-shell formation process. The ferrous ascorbate salt and folic acid have been converted into different iron oxides belonging to different crystal systems. Since all the compounds are iron-based, it can be inferred that the entire surface of the TiO₂ NPs has been coated and the thickness of the coating is also more than 10 nm. X-rays can easily penetrate the 10 nm coating thereby displaying the structural peaks of the core beneath. But in this case, no TiO₂ phase has been obtained which only indicates that the thickness of the iron coating is > 10 nm. In our previous study [13], the thickness of the shell was ~ 10 nm and the XRD pattern of the Autrin tablet-coated anatase TiO₂ NPs was exactly similar to that of the bare TiO₂ NPs which was possible only because of the thin shell formation. The XRD pattern of the P is almost similar to that of the F but a little less crystalline. It can be concluded that the PEG layer lowers the crystallinity of the sample.

Table 1

The symbols used in this report and their meanings.
Symbol	Meaning
D	Imatinib
FXT	FericipXT (Iron Supplement)
T	Rutile TiO₂ NPs
F	FericipXT@ rutile-TiO₂ NPs (Iron tablet-coated rutile TiO₂ NPs)
P	PEGylated FericipXT@ rutile-TiO₂ NPs (PEG coating onto the iron tablet-coated TiO₂ NPs)
TD	Imatinib (Drug) loaded onto rutile-TiO₂ NPs
FD	Imatinib (Drug) loaded onto FericipXT@rutile-TiO₂ NPs
PD	Imatinib (Drug) loaded onto PEGylated FericipXT@rutile-TiO₂ NPs

Table 2

Different phases obtained after XRD analysis of FericipXT-coated rutile TiO₂ NPs with 1:1 core:shell ratio and their quantification.
JCPDS Pattern	Compound Name	Quantification	Crystal System
01-084-0311	Iron (III) oxide- alpha (Fe₂O₃)	8.9%	Rhombohedral
01-089-2428	Dioxygen-alpha (O₂)	17.8%	Monoclinic
01-089-0599	(Hematite) Iron (III) oxide-alpha (Fe₂O₃)	5%	Rhombohedral
01-076-1821	Iron (III) oxide-beta (Fe₂O₃)	24.8%	Hexagonal
01-086-1350	Iron Oxide (Fe_2.937O₄)	1%	Cubic
01-080-2186	Iron oxide-gamma (Fe_21.34O₃₂)	19.8%	Tetragonal
01-089-5894	(Maghemite Q Iron Oxide) Iron (III) oxide-gamma (Fe_1.966O_2.963)	22.7%	Tetragonal

The XRD plot of D shows a set of diffraction peaks between 10^o and 30^o. The XRD plot of FericipXT doesn’t show any well-defined peaks, rather an almost flat plot was obtained indicating the amorphous nature of the tablet. The XRD pattern of P was similar to F with a few extra peaks of low intensity emerging at 24.686^o, 28.918^o and 29.726^o which correspond to iron titanium oxide (Fe₉TiO₁₅) and orthorhombic phase of Fe₂O₃. Antarnusa et al. [14] coated Fe₃O₄ NPs with PEG 4000 and observed the appearance of extra phases corresponding to α-Fe₂O₃, α-FeO(OH) and γ-FeO(OH). The XRD plots marked as TD, FD and PD have retained the diffraction pattern obtained for the host NPs. The drug loading has not disturbed the diffraction pattern of the host NPs and thus, the diffraction peaks of the individual NPs are retained. However, no diffraction peaks related to Imatinib have been detected in the individual XRD patterns of TD, FD and PD. This implies that Imatinib is present in an amorphous state onto the NPs. Likewise, Li et al. [15] synthesized drug, 5-FU loaded chitosan-coated ZnSe/ZnS NPs (5-Fu–CS–ZnSe/ZnS NPs) and observed that XRD plot of 5-FU displayed diffraction peaks indicating its crystalline nature, however, no such peak of the drug was obtained in the XRD of 5-Fu–CS–ZnSe/ZnS NPs depicting that the drug was present in its amorphous form onto the functionalized NPs. Raj et al. [16] prepared solid-lipid NPs and loaded them with cytarabine, an anticancer drug meant for treating leukemia. The XRD plot obtained for cytarabine was crystalline in nature, but the XRD pattern of cytarabine-loaded solid-lipid NPs didn’t show the characteristic peak of cytarabine. They too reported that cytarabine was present in its amorphous form onto the NPs. In another study, the XRD of quercetin presented sharp diffraction peaks confirming its crystalline nature but when the same quercetin was loaded onto PEGylated CdSe/CdS and PEGylated CdSe/ZnS core/shell NPs and their XRD characterization was done, no peak of the antioxidant molecule was detected. This confirmed that quercetin existed in non-crystalline i.e. amorphous state onto the NPs [17]. Patel et al. [18] suggested that the crystalline behaviour of the conjugant is often suppressed when they are loaded onto the NPs resulting in an amorphous form.

FT-IR Spectroscopy

Figure 2 displays the FTIR spectra obtained for different samples and Table 3 highlights the different functional groups associated with the samples corresponding to the peaks obtained in their relevant FTIR spectra. In case of Imatinib (D), the broad region between 3200–4000 cm^− 1 shows characteristic peaks at 3237.05 cm^− 1, 3414.83 cm^− 1, 3475.74 cm^− 1 and 3550.83 cm^− 1 corresponding to the stretching vibration of the O-H group [19]. A pattern similar to D can be observed in TD, FD and PD for the said range of wavenumbers which confirms the presence of the drug onto these NPs along with the presence of stretching mode of the O-H group. The peak corresponding to 3550.83 cm^− 1 in D has been observed at 3546.26 cm^− 1 in PD. The peak at 3475.74 cm^− 1 in D has been shifted to 3463.61 cm^− 1, 3463.83 cm^− 1, 3462.55 cm^− 1, 3470.46 cm^− 1 in T, P, TD and PD, respectively. For a peak at 3414.83 cm^− 1 in D, the corresponding peak in FXT, T, F, P, TD, FD, PD has been obtained at 3429.14 cm^− 1, 3425.40 cm^− 1, 3434.15 cm^− 1, 3414.61 cm^− 1, 3431.66 cm^− 1, 3416.92 cm^− 1, 3414.12 cm^− 1, respectively. The peak obtained at 3237.05 cm^− 1 in D has only been observed in PD at 3238.17 cm^− 1. For the same range of wavenumbers, FericipXT showed a broad region which was also obtained in F. With this, we can confirm the successful coating of rutile TiO₂ NPs with FericipXT. The FTIR patterns of T-TD, F-FD and P-PD also match as T, F and P represent the host NPs which after loading of the drug became TD, FD and PD, respectively.

D exhibits a small peak at 2920.87 cm^− 1 which corresponds to the stretching mode of the C-H group. The same peak has been obtained in FD and PD at 2917.30 cm^− 1, 2915.98 cm^− 1, respectively. D showed a double-peak pattern at 1617.31 cm^− 1 and 1638.36 cm^− 1, respectively corresponding to C = C stretching. The similar pattern was repeated in TD, FD and PD at (1622.01 cm^− 1 & 1637.07 cm^− 1), (1619.09 cm^− 1 & 1637.31 cm^− 1) and (1617.73 cm^− 1 & 1637.72 cm^− 1), respectively. This double-peak pattern was observed only in the drug-loaded NPs and was not observed in the bare NPs. Instead, the bare NPs such as T, F and P demonstrated a single peak at 1636.22 cm^− 1, 1634.89 cm^− 1, 1636.59 cm^− 1, respectively corresponding to C = C stretching.

Table 3

A description of wavenumbers relating to the FTIR spectra and their corresponding functional groups for all the samples.
S.No.	Wavenumber (cm^− 1)	Sample ID (in sequence of the wavenumbers mentioned)	Functional Groups
1	3550.83, 3546.26	D, PD	Stretching vibration of the hydroxyl group O-H
2	3475.74, 3463.61, 3463.83, 3462.55, 3470.46	D, T, P, TD, PD	Dimeric OH stretch
3	3414.83, 3429.14, 3425.40, 3434.15, 3414.61, 3431.66, 3416.92, 3414.12	D, FXT, T, F, P, TD, FD, PD	H-bonded OH stretch
4	3237.05, 3238.17	D, PD	Normal ‘‘polymeric’’ OH stretch
5	2920.87, 2917.30, 2915.98	D, FD, PD	Methylene C-H asym./sym. stretch
6	2879.65	FD	Methylene C-H asym./sym. stretch
7	1776.19	FD	C = O stretching
8	1617.31, 1638.36; 1622.01,1637.07;1619.09, 1637.31; 1617.73, 1637.72;	D, TD, FD, PD	C = C stretching
9	1620.90, 1636.22, 1634.89, 1636.59	FXT, T, F, P	C = C stretching
10	1466.85, 1452.94, 1449.61, 1462.97, 1452.30	D, F, TD, FD, PD	C-H bending
11	1420.13, 1425.66, 1418.94	D, P, TD	C-H bending
12	1386.88, 1381.78, 1382.41, 1381.65, 1383.43, 1383.00, 1383.85	D, FXT, T, P, TD, FD, PD	C-H bending
13	1351.99	FD	O-H bending
14	1312.90	D	O-H bending
15	1282.75, 1297.97, 1288.37	D, FD, PD	C-O stretching
16	1250.80	FD	C-O stretching
17	1223.37	D	C-O stretching
18	1167.39, 1157.86, 1156.93	D, P, TD	Hydrate
19	1117.03, 1122.51, 1124.66, 1109.98, 1109.59, 1110.41	FXT, T, F, P, TD, PD	Secondary or tertiary alcohol C-O stretching
20	1035.04, 1015.35, 1029.75, 1056.21, 1029.96, 1097.75, 1058.29	D, F, P, TD, FD, PD	C-N stretching
21	903.50, 916.42, 945.98	F, P, FD	C‒H (Bending of aromatic hydrocarbons)
22	803.72, 876.80, 878.54, 897.28	D, FXT, F, PD	C‒H (out of plane bending of aromatic hydrocarbons)
23	753.96	D	C‒H (Bending of aromatic hydrocarbons)
24	620.10, 617.44, 613.00, 619.31, 613.76, 621.13, 617.87, 620.01	D, FXT, T, F, P, TD, FD, PD	Ti-O-Ti bridging stretching mode, Fe-O in case of FXT
25	525.57, 507.80	TD, FD	Bending vibrations of O-Ti-O
26	480.34, 488.83, 476.60, 480.83	D, FXT, FD, PD	O–Ti–O bonding

The peaks obtained between 1380–1467 cm^− 1 contribute to C-H bending and those obtained in the range 1100–1280 cm^− 1 belong to C-O stretching. The peaks obtained between 750–900 cm^− 1 correspond to out-of-plane bending of C-H aromatic hydrocarbons. The peak at 613 cm^− 1 in T is attributed to Ti-O-Ti bridging stretching mode [10]. Moreover, the peak obtained at 620.10 cm^− 1, 619.31 cm^− 1, 613.76 cm^− 1, 621.13 cm^− 1, 617.87 cm^− 1 and 620.01 cm^− 1 corresponds to Ti-O-Ti bridging stretching mode of D, T, F, P, TD, FD and PD, respectively. The reason that the peak at 620.10 cm^− 1 in D is related to Ti-O-Ti bonding is due to the fact that an Imatinib tablet also has a coating of Titanium dioxide. However, the peak obtained at 617.44 cm^− 1 in FXT can be attributed to the Fe-O bond [20]. The FTIR results validate that Imatinib was conjugated over T, F and P.

UV-Vis Spectroscopy

In order to find the percent in-vitro drug release by measuring the absorbance of the drug (Imatinib) released, at the first, the calibration curve of Imatinib was plotted in different pH mediums, viz. pH 4.4, 7.4 and 9.0 in which the drug-release study was to be conducted. The calibration curve was plotted with the concentration of Imatinib in µg/ml along the x-axis and the absorbance value obtained along the y-axis. For this, the stock solution of Imatinib was prepared under three different pH environments. The absorption spectra of the drug in three different buffer solutions against known values of concentration were acquired and plotted. Figure 3(a), 3(b) and 3(c) denote the calibration curve of the drug (Imatinib) obtained under pH 4.4, 7.4 and 9.0, respectively. With the help of the equation of straight line and least square fitting, the slope and intercept for all the three categories were obtained. These slopes and intercepts were used to calculate the concentration of the drug released in the in-vitro drug-release study.

These curves have been obtained by plotting the concentration of the drug (µg/ml) added along the x-axis and the corresponding absorbance measured along the y-axis. The absorbance of the drug increased with increasing concentration. Imatinib and mostly the maximum percentage of chemotherapeutic drugs exhibit maximum solubility in acid which decreases with increasing pH. This can be verified from the calibration curves obtained below as the maximum value of absorbance received for 1000 µg/ml is the highest under pH 4.4 and the least in the pH 9.0. Figure 4(a), 4(b) and 4(c) denote the UV-Visible absorbance plots obtained for the D, T, F and P under pH 4.4, 7.4 and 9.0, respectively. The drug showed the maximum absorbance in all the pH conditions. In pH 4.4 and 7.4, TD and FD showed almost similar behaviour with them showing more absorption in acidic medium and very less absorption in the neutral medium. Furthermore, PD showed a very limited absorption in acidic medium and even lesser in neutral medium. But the absorbance displayed by PD in neutral medium was more than TD and FD which can be attributed to the biocompatible nature of the PEGylated NPs. Overall, the absorbance behaviour demonstrated by TD, FD and PD ascertains their usage in pH-responsive drug delivery applications.

HR-TEM

Figure 5(a) depicts the HR-TEM image of T. They belong to the size range 25–35 nm. Figure 5(b) displays the image of F. Their size is approximately 60 nm. Thus, the iron coating has augmented the size of the core NPs. Further the PEGylation of the NPs has further increased the size of the resulting NPs to the range 120–145 nm. Figure 5(c) shows the HR-TEM image of P. Both the iron tablet-coating and the PEG layer can be easily observed as the distinct layers from the image.

In addition, the morphology of all the NPs in Fig. 5(a), 5(b) and 5(c) are almost spherical. Liu et al. [21] observed a slight increase in the size of the TiO₂ NPs when they were functionalized with hyaluronic acid. The HRTEM of D is shown in Fig. 5(d) and that of TD is shown in Fig. 5(e). The characteristic patterns obtained for D in Fig. 5(d) can also be seen in Fig. 5(e) ensuring the loading of the drug onto the NPs. The inset in Fig. 5(e) explains that the shape of the particles after loading is rounded rectangle-like. The longer side of the rectangular shaped TD varied between 40–75 nm. Clearly, there has been an increase in size as well as morphological change. Figure 5(f) demonstrates the HRTEM image of FD. It is obvious that the drug has been loaded onto the NPs as the NPs themselves were independent spherical shaped particles as depicted in Fig. 5(b). However, now the particles have been aggregated. Further, one can observe the elongation of the NPs as well. The inset of Fig. 5(f) shows the magnified image of the NPs. The layer around the NPs is clearly visible. The dark patches can be associated with the loaded drug. The nanostructures obtained are highly agglomerated due to which their size cannot be computed by analyzing the image. The HRTEM image of PD is shown in Fig. 5(g) with the inset showing its magnified view. These NPs have somehow managed to retain their round shape, although they are not perfectly round. The layer around the NPs can be seen. However, the particle size post drug-loading has seemed to decrease. PD seem to be agglomerated and possess a varied size distribution in the range 30–80 nm. This reduction in size of PD might be due to the lattice strain generated on account of loading of Imatinib molecules which have restricted the growth and nucleation of the P during the drug-loading process [22].

SAED

Figure 6(a), 6(b) and 6(c) present the SAED patterns for T, F and P, respectively. The SAED pattern for T shows very bright circular concentric dotted patterns which is indicative of the highly crystalline nature of the NPs as shown in Fig. 6(a). Figure 6(b) displays diffuse concentric rings which show that the crystalline behaviour of T has weakened after the coating with the iron tablet. Finally, Fig. 6(c) again displays a scattered dotted pattern which is indicative of the crystalline nature of P. A few random spots are visible due to the PEG coating on the NPs. This implies that the crystallinity has decreased due to polymer coating and defect density has increased [23]. These observations get along well with the results obtained from XRD analysis. Gayathri et al. [24] coated Eu:Gd₂O₃ NPs with silica and a diffuse ring pattern was obtained which confirmed the amorphous nature of the NPs. Initially, a well-defined ring structure was obtained in the SAED pattern for the Eu:Gd₂O₃ NPs.

DLS and ZP

DLS is an ideal technique for performing size distribution measurements. DLS studies more than tens of thousands of particles thereby minimizing the error generated due to single particle encounters. The hydrodynamic size and Polydispersity Index (PDI) are both crucial parameters to optimize the performance and understand the in-vitro migration of the NPs. The readings were taken by dispersing the NPs in water where 5 runs of each sample were taken for 30 seconds each. The measurements were performed at 25^oC using a wavelength of 658.0 nm. The difference between hydrodynamic diameter and core diameter is that core diameter tells how much drug can be put inside the NPs whereas hydrodynamic diameter is equivalent to the diameter of the particle which would experience the same drag force in the fluid as would the dispersed nanoparticle in question. Hydrodynamic diameter comes into the picture when NPs are dispersed into some liquid medium. When NPs are dispersed in a liquid, the hydration layer gets attached over their surface due to which the apparent size of the NPs increases. Figure 7(a) illustrates the intensity-weighted size distribution for T, F, P, TD, FD and PD, respectively. The data is plotted between differential number G(d) and the diameter (in nm). The mean diameters obtained for T, F and P are 449.1 nm, 438.2 nm and 438.5 nm, respectively. The mean diameters obtained for TD, FD and PD are 198.3 nm, 59 nm and 432.2 nm, respectively. For T, a straight line plot was obtained. Also, a close to zero value of PDI, i.e. 0.005 confirms that there is least width in the distribution plot for T. A little decline in mean diameter has been observed as T was coated with an iron supplement. Not much difference has been observed in the mean diameters of F and P. A drastic decrease in the mean diameter has been observed when both T and F were loaded with Imatinib. Thus, drug loading has modified the mean diameter of both T and F, however, PEGylated NPs didn’t face much difference post drug-loading. For FD, a very narrow size distribution has been obtained with the peak value of 59 nm. This observation matches with our HR-TEM result which confirms the size of FericipXT-coated rutile TiO₂ NPs to be ~ 60 nm. For the rest of the samples, the obtained size is more than what obtained from HR-TEM. The intensity-weighted mean diameters are different from hydrodynamic diameters of the NPs. Table 4 summarises the hydrodynamic diameter in nm and PDI of T, F, P, TD, FD and PD. The PDI of all the samples was below 0.3 which indicates a narrow size distribution for all the NPs. Moreover, a non-zero value of PDI is an indicator of width in a distribution. When dispersed NPs move through a fluid, a thin electric dipole layer of the solvent attaches to their surface. This layer affects the movement of the NPs through the medium. Thus, hydrodynamic diameter is the diameter comprising the core, shell, surface coating (if any) plus the solvent layer, whereas, in analysis via HR-TEM, the so-called solvent or the hydration layer is not present. In addition, TEM is a number-based technique whereas DLS is an intensity-based technique [25][26]. Hence, it is evident that hydrodynamic size is greater than the size obtained via measurements like Debye-Scherrer’s formula, FESEM and HR-TEM. In the present study, the sizes of the NPs are much greater than those predicted from the HR-TEM study which can be attributed to the hydrated layer present around them. However, the sizes of the NPs and their PDI obtained are acceptable and are indicative of appropriate synthesis of the NPs.

Table 4

Hydrodynamic diameter and Polydispersity Index of T, F, P, TD, FD and PD.
S.No.	Symbol	Hydrodynamic Diameter (nm)	Polydispersity
1	T	466.0 nm	0.005
2	F	449.5 nm	0.227
3	P	642.2 nm	0.277
4	TD	394.5 nm	0.252
5	FD	437.4 nm	0.198
6	PD	557.2 nm	0.241

Zeta Potential is basically the charge which develops at the interface between a solid surface and the liquid medium. The motion of the particles is a product of Brownian motion and the attraction or repulsion occurring among the particles under the influence of an electric field. In case the repulsive force dominates over the attractive force, the formulation becomes stable. The values of ZP indicate the interaction of the NPs with the surroundings and can be used to understand their stability, surface characteristics and adsorption phenomena. The values of zeta potential greater than + 25 mV or less than − 25 mV provide more stability to the sample as the particles will stay dispersed for longer duration, dominating the vander waals forces of attraction. Figure 7(b) illustrates the ZP plots for T, F, P, TD, FD and PD, respectively. The mean zeta potential obtained for T, F, P, TD, FD, PD are − 17.21 mV, + 4.19 mV, -32.89 mV, + 2.30 mV, -7.53 mV and − 16.42 mV, respectively. ZP is known to vary with pH and gets more positive or negative with acidic and basic pH, respectively. ZP neither measures the charge nor the charge density, rather it tells about the surface potential. Thus, only the magnitude of ZP matters rather than the robust positive/negative charges associated with it [27]. Here, PEGylated NPs have shown the maximum stability with − 32.89 mV as the value of zeta potential. Thus, it is confirmed that PEGylation is beneficial for enhancing the stability of the NPs which can then be effectively used in drug-delivery applications.

VSM

Undoped TiO₂ NPs also demonstrated weak ferromagnetism with 0.004 emu/g saturation magnetization as shown in Fig. 8(a). Figure 8(b) illustrates the VSM plot of FericipXT, F and FD. The plots of FericipXT and FD are almost superimposed. The most striking observation was that the magnetization induced in F was much greater than that induced in the FericipXT alone. However, the magnetization induced in F was again reduced when it was loaded with D. In addition, the saturation magnetization of P was lower than F. Similar observation made by Tai et al. [28] was that the saturation magnetization of the magnetic NPs decreased when they were coated with PEG 600 Da and the value was further decreased with the increase in PEG content. A similar study conducted also concluded that the saturation magnetization of the magnetite NPs decreased with increasing PEG content [14]. Further, Fig. 8(c) shows the VSM plots of P and PD, respectively. Near superparamagnetic behaviour was observed for both the samples. However, the magnetization induced in PD was more than that in P. Figure 8(d) compares the VSM plots of FericipXT, FD and PD and all the three plots seem to be nearly overlapping. Thus, PD can be suitably used for the magnetically-stimulated drug delivery applications. Liu et al. [29] synthesized TiO₂ coated Fe₃O₄ nanospheres for the delivery of Daunomycin. They observed that the coating of TiO₂ onto the superparamagnetic Fe₃O₄ resulted in a superparamagnetic VSM plot for the core-shell nanospheres so obtained but with reduced saturation magnetization which was further reduced upon the loading of daunomycin. Thus, the drug-loading often decreases the saturation magnetization of the otherwise magnetic compound. But here we have observed that the magnetic behaviour of T has been significantly improved upon coating with FericipXT and upon PEGylation. These NPs offer notable potential to be used as magnetically-guided drug-delivery vehicles.

In-vitro drug release study

For the drug release studies, we have investigated the release behavior under different pH levels. Various parameters such as the chemistry of the drug, drug-NPs interaction, method of drug loading, percent of drug loading, solubility of the drug in the release media, the surface modification and functionalization affect the rate of drug release under different conditions [30]. The following terminology has been used to indicate the samples in different pH solutions as shown in Table 5.

Table 5

The terminologies used for studying drug release from different samples under different pH levels.
pH → Samples ↓	pH 4.4	pH 7.4	pH 9.0
TD	T4	T7	T9
FD	F4	F7	F9
PD	P4	P7	P9

For all the pH mediums, the study was undertaken for 2000 minutes. The dialysis tube chosen for the study also influences the rapidity or retardation of the drug release. Figure 9 shows the drug release profile from the samples T4, F4 and P4 at pH 4.4 with cumulative release percentage plotted along the y-axis and the time (in minutes) along the x-axis. The inset displays the drug release plot obtained for the first two hours. The pH around malignant tumors is mostly acidic, that’s why most of the chemo drugs are designed to release faster around acidic environments. The orally administered drugs first reach into the stomach after consumption, where the highly acidic medium breaks them down into their components from where the drug-molecules release into the bloodstream. For the acidic pH, out of all the three samples, P4 showed much controlled drug release behaviour as it released only 23.29% of the drug in the first two hours, whereas T4 and F4 exhibited initial burst release behaviour by releasing 50.9% and 51.76% of the drug, respectively in just 120 minutes. Thus, half of the drug was released by both T4 and F4 at the end of two hours. The drug release profile of both T4 and F4 almost matched for the entire release pattern and both released approximately 99% of the drug in the study of 2000 minutes. P4 demonstrated a very controlled drug-release behaviour. At the end of 1350 minutes, P4 also showed 96% of the drug release like the other two and from then onwards, its profile also overlapped with those of T4 and F4. Thus, in acidic medium, P proved to be the best drug-carrier showcasing a very controlled drug release. Moreover, the plot obtained for P4 exhibited no sharp ups or downs, rather it showed a very smooth linear increment for the total time period of the study. Here, we can conclude that PEGylation of the NPs improves the sustained drug release behaviour of the NPs by ensuring that the same desired quantity of the drug will be released systematically for longer duration. This enhances the drug retention but minimizes the side-effects, quantity of dosages and drug release at unwanted sites.

Likewise, Fig. 10 demonstrates the drug release profile of T7, F7 and P7 at pH 7.4. The pH of normal blood generally lies between the range 7.35 to 7.45. The NPs to be used as drug carrying vehicles are expected to release the drug solely under acidic environments and less to no release under normal pH. With this minimal drug release can be assured during the circulation and more effective drug release at the target locations can be achieved. Under normal pH, T7 showed the maximum cumulative release percentage of 31.89% whereas F7 released 14.86% and P7 released 18.64% of the drug respectively. Both F7 and P7 showed overly controlled drug release behaviour. The plots of both F7 and P7 ran parallely, however, F7 outperformed P7 in the context of least drug released under normal pH. The inset in Fig. 2 shows the initial release pattern in the first two hours. The better performance of F7 and P7 than T7 concludes that the surface modification of T improves their drug-release behaviour. It is clear from the observation that iron coating and the PEG layer are better able to hold the drug effectively for longer hours, mitigating the chances of unnecessary drug release before reaching the target site. Hettiarachchi et al. [31] have reported that the pH and redox-triggered release of doxorubicin (DOX) from carbon dots was faster in acidic pH than in pH 7.4. Xu et al. [32] reported in their study that more than 90% of the bare imatinib was released in the pH 5.5 in the initial 8 hours. However, when imatinib was loaded onto the liposomes, only 48.3% of the drug was released in 48 hours. PEG molecules are known to retain their helical structure in aqueous solutions [33]. In the present study, the PEGylated NPs showed the most sustained drug release behaviour than T and F under acidic and neutral pH conditions. Kumskova et al. [34] reported that polymers of different molecular weights affect the different parameters of the drug-loaded NPs such as percent drug loading, pattern of drug release and degradation of the NPs. Jain et al. [25] reported initial burst release of DOX from PEGylated CdSe/ZnS core-shell NPs in the first 180 minutes of the drug-release study followed by slower and sustained release of the drug. The drug release was higher in the acidic pH than in the neutral pH. The drug-release mechanism was found to be associated with Weibull with a lag-time model which indicates the release of the drug by dissolution. Bharati et al. [26] found in their study that PEG-diamine functionalized CdSe/ZnS NPs are capable of regulating the pH-dependent in-vitro release of quercetin. An initial burst release took place for the first 200 minutes and more quercetin was released at neutral pH than at mildly acidic pH. Thus, different drug molecules favour different pH conditions to get released.

Additionally, the samples were also tested under the basic medium of pH 9.0. Figure 11 illustrates the drug-release profiles for T9, F9 and P9. The inset shows the release profile for the initial two hours. F9 almost restricted the release of the drug and showed only 4.73% of the drug-release in the entire study. However, P9 attained 19.1% of the drug release and T9 released 17.44% of the drug. This observation was strikingly different as P9 behaved strangely under this pH level. Normally, as the pH increases, the drug-release should decrease. TD followed this notion and they released 99% of the drug at pH 4.4, 31.89% of the drug at pH 7.4 and 17.44% of the drug at pH 9.4. Similarly, FD released 99% of the drug under pH 4.4, 14.86% of the drug under pH 7.4 and just 4.73% of the drug at pH 9.4. Both TD and FD demonstrated maximum drug release under acidic conditions and accordingly limited the release of the drug as the pH was increased to neutral with further restricting the drug release when the pH turned basic. P outperformed the other two under acidic conditions by exhibiting sustained drug release by still achieving 99% of the drug-release in the end. In neutral pH, the cumulative drug-release achieved was reduced by 80.36%. But in the buffer solution exhibiting basicity, PD showed 19.1% of the drug release which is more than that achieved in neutral pH by 0.46%. In other words, it can be put as the P behaved alike under both neutral and basic pH mediums. It was expected that the cumulative drug release percentage will be lesser in basic pH than that acquired in neutral pH, but no such observation was made. Similar behaviour was observed by Jain et al. [35] where the swelling rate of Polymethyl Methacrylate (PMMA) accelerated in a slightly alkaline medium on account of hydrolytic cleavage of the polymer chains. The hydrolytic cleavage results in acidic species in the polymer network creating repulsion among positively charged polymer chains thereby increasing the swelling ratio.

Kadivar et al. [36] in their study reported that 99.56% of the Imatinib gets released within 20 minutes in 0.1 N HCl solution. The NPs synthesized by us demonstrated sustained drug-release behaviour which is much needed for an effective chemotherapeutic treatment. If a drug is retained for a longer time, this would diminish the requirement of drug dosages thereby minimizing the side-effects associated with drug-overdose. The maximum drug release attained by the NPs synthesized by us under acidic pH conditions suggests that they undergo comparatively faster decomposition and promote faster drug-release under acidic pH conditions as compared to neutral pH conditions. In nutshell, the results revealed that the release of Imatinib is strongly pH-responsive. The pH-dependent release behaviour of Imatinib can help improve the efficiency of the drug-delivery system involving the nanocarriers and the drug.

Hemolysis Assay

Hemolysis assay was performed to understand the blood compatibility of the P. PEGylation is performed onto the NPs to improve their biocompatibility and the same can be observed from the results depicted in Fig. 12(a). 0% hemolysis was observed in the blood sample obtained by centrifuging it with 0.9% saline solution and this was taken as the negative control. 100% hemolysis was obtained by centrifuging the blood sample with 1% Triton X-100 solution and this was treated as the positive control. The ratio of hemolysis of the samples was in the range between 0-1.2% which was less than the critical safe limit of hemolysis for biomaterials as per ISO/TR 7406. Thus, the damage posed by the samples was very little. The least hemolysis occurred in the samples 2 to 5 was almost undetectable by naked eyes. Moreover, it was observed that the percentage of hemolysis was incremented slightly with increase in the concentration of the NPs.

The actual pictures of the samples 2 to 5, positive control and negative control after centrifugation are depicted in Fig. 12(b). The percentage of hemolysis increased slightly with the increase in the concentration of the NPs. Aisaka et al. [37] suggested that grave attention should be paid towards the polymorphs of the NPs when studying their toxicity. However, in their experiment, no direct evidence of generation of any oxidative stress intracellularly by TiO₂ NPs was observed. The plasma abolished any sort of hemolysis initiated by both anatase and rutile TiO₂ NPs. Thus, our PEGylated FericipXT-coated rutile TiO₂ NPs are a safer option for drug-delivery applications. Additionally, there is evidence which confirms that PEGylation minimizes the hemolysis induced by NPs and also the percent hemolysis is significantly reduced by increasing the PEG content. PEG coating ameliorates the systemic delivery of the therapeutic and is also helpful in overcoming extracellular barriers. Further, the biodistribution of the NPs and their clearance from the body is also promoted [38].

Mathematical Models and Understanding The Drug Release Mechanism

In order to understand the drug-release mechanism, the in-vitro drug-release data was studied using different mathematical models. The different models proposed assist in understanding the dissolution profile of the drug with respect to time. Different factors such as size of the NPs, their shape, porosity, crystallinity, surfactants, polymer-coating, the drug itself and the process of drug-loading affect the release kinetics. The drug releases in various ways such as burst release, extended release, controlled release, delayed release and sustained release [39]. The first 60% of the release curve obtained is employed for the statistical analysis which tells about the mechanism governing the drug-release pattern. The drug-release profiles obtained for Imatinib release from different samples under different pH levels were analyzed against various mathematical models such as Zero order model, First order model, Higuchi model, Hixson-Crowell model, Weibull model, Noyes-Whitney model, Korsmeyer-Peppas model, Peppas-Sahlin model and 2nd degree polynomial model.

Table 6. Representation of R² values obtained after fitting the data against different models.

Samples >>	F4	T4	P4	F7	T7	P7	F9	T9	P9
pH	4.4			7.4			9.0
Zero Order Model	0.7129	0.7323	0.9807	0.8166	0.8692	0.7345	0.7475	0.2305	0.3384
First Order Model	0.9094	0.9138	0.9655	0.8388	0.8910	0.7647	0.7590	0.2617	0.3770
Higuchi Model	0.9178	0.9178	0.9178	0.9178	0.9178	0.9178	0.9178	0.9178	0.9178
Hixson-Crowell Model	0.8763	0.8887	0.9894	0.8316	0.8840	0.7548	0.7552	0.2512	0.3639
Weibull Model	0.9533	0.9464	0.9924	0.9682	0.9777	0.9752	0.9805	0.8467	0.8520
Noyes-Whitney Model	0.9094	0.9138	0.9655	0.8388	0.8910	0.7647	0.7590	0.2617	0.3770
Korsmeyer-Peppas	0.9895	0.9950	0.9933	0.9866	0.9887	0.9932	0.9926	0.9953	0.9662
Peppas-Sahlin	0.9895	0.9950	0.9933	0.9876	0.9894	0.9939	0.9928	0.9953	0.9662
2^nd Degree Polynomial	0.9180	0.9570	0.9920	0.8520	0.9800	0.9340	0.8350	0.7620	0.8630

The model providing the highest value of coefficient of determination R² will be chosen as the model describing the drug-release mechanism. Table 6 describes the coefficient of correlation R² values for all the samples against nine models chosen by us. Among all the models, the highest value of R² is obtained for the Peppas-Sahlin model. Peppas and Sahlin [40] fabricated a release kinetics model given by Eq. (6) as follows:

\(\frac{{M}_{t}}{{M}_{\infty }}\) = K₁t^m + K₂t^2m -------------(6)

where K₁, K₂ and m are constants. The highest R² values denote that the drug-release mechanism can be easily understood by means of the Peppas-Sahlin model which suggests that the release of the drug from the system is governed by a combination of Fickian diffusion and Case II relaxation. Fickian diffusion release is due to the general molecular diffusion of the drug which occurs because of the chemical potential gradient. Case II relaxation release is associated with the stress and state-transition occurring in hydrophilic polymers which swell in water or biological fluids. Thus, we can conclude that the release of Imatinib from the different NPs-based formulations is best fitted to the Peppas-Sahlin model and the drug follows Fickian diffusion coupled with Case II relaxation.

Rutile TiO₂ NPs were coated with iron supplement, FericipXT to enhance their magnetic response for magnetically guided drug delivery application. They were further PEGylated to improve their biocompatibility and reduce the toxicity. The formulations synthesized were tested for in-vitro delivery of Imatinib, a chemotherapeutic in three different pH levels. A sustained release of the drug was achieved by the PEGylated NPs. Moreover, different nano-formulations were observed to be releasing the drug following the Peppas-Sahlin model under all the pH conditions. Thus, the drug release mechanism followed both Fickian diffusion and Case II relaxation. These drug carrying NPs offer potential applications in magnetically-guided targeted delivery of chemotherapeutics as confirmed from the findings of VSM characterization. The chances of premature drug release can be diminished and those of site-specific drug release can be greatly enhanced thereby minimizing the side effects of the dosages provided. Moreover, the PEGylated NPs appeared non-toxic as the percent hemolysis occurred belonged to a safe level.

Conflict of Interest

This statement is to certify that all authors have seen and approved the manuscript being submitted. We warrant that the article is the Authors' original work. We warrant that the article has not received prior publication and is not under consideration for publication elsewhere. The authors have no Conflicts of Interest to disclose.

Author Contributions

The authors confirm their contribution to the paper as follows:

Conceptualization - S. Bhullar
Data curation - S. Bhullar
Formal Analysis - S. Bhullar , N. Goyal , S. Gupta
Funding acquisition - S. Bhullar , S. Gupta
Investigation - S. Bhullar
Methodology - S. Bhullar , S. Gupta
Project administration - S. Bhullar , S. Gupta
Resources - S. Bhullar , N. Goyal , S. Gupta
Software - S. Bhullar , N. Goyal
Supervision - N. Goyal , S. Gupta
Validation - S. Bhullar , N. Goyal , S. Gupta
Visualization - S. Bhullar
Writing – original draft - S. Bhullar
Writing – review & editing - S. Bhullar , N. Goyal , S. Gupta

All authors reviewed the results and approved the final version of the manuscript.

Ethics Approval and Consent to Participate

Not applicable

Consent for Publication

Not applicable

Funding
The authors declare that (s)he has no relevant or material financial interests that relate to the research described in this paper.

Acknowledgement

We are grateful to the Sophisticated Analytical Instrumentation Facility (SAIF) and CIL, Panjab University, Chandigarh for timely characterizations.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

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FericipXT-Coated PEGylated Rutile TiO2 Nanoparticles in Drug Delivery: In-vitro Assessment of Imatinib Release

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Version 1

Abstract

Figures

1 Introduction

2 Experimental Section

2.1 Materials

2.2 Methods

2.2.1 Synthesis of PEGylated Iron Supplement-Coated Rutile TiO₂ NPs

2.2.2 Synthesis of Drug-Loaded PEG/Fe@TiO₂ NPs

2.2.3 In-Vitro Drug Release by Dialysis Method

2.2.4 Hemolysis Assay

2.3 Characterization

3 Results and Discussion

4 Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

FericipXT-Coated PEGylated Rutile TiO2 Nanoparticles in Drug Delivery: In-vitro Assessment of Imatinib Release

Status:

Version 1

Abstract

Figures

1 Introduction

2 Experimental Section

2.1 Materials

2.2 Methods

2.2.1 Synthesis of PEGylated Iron Supplement-Coated Rutile TiO2 NPs

2.2.2 Synthesis of Drug-Loaded PEG/Fe@TiO2 NPs

2.2.3 In-Vitro Drug Release by Dialysis Method

2.2.4 Hemolysis Assay

2.3 Characterization

3 Results and Discussion

4 Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

2.2.1 Synthesis of PEGylated Iron Supplement-Coated Rutile TiO₂ NPs

2.2.2 Synthesis of Drug-Loaded PEG/Fe@TiO₂ NPs