Novel Cytotoxicity Study of Strontium (Sr) Doped Iron Oxide (Fe3O4) Nanoparticles Aided with Ibuprofen for Drug Delivery Applications

doi:10.21203/rs.3.rs-2908876/v1

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Research Article

Novel Cytotoxicity Study of Strontium (Sr) Doped Iron Oxide (Fe₃O₄) Nanoparticles Aided with Ibuprofen for Drug Delivery Applications

https://doi.org/10.21203/rs.3.rs-2908876/v1

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Journal Publication

published 04 Jul, 2023

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Purpose

This work aimed to study the drug delivery applications of iron oxide (Fe₃O₄) nanoparticles with strontium (Sr) doping with varying molar ratios prepared by the co-precipitation route. The impact of increased strontium content on the particle size and magnetic properties was investigated. The impending of these nanoparticles for drug loading, drug release, and their respective cytotoxicity was also inspected.

Methods

First, iron oxide nanoparticles were doped with various amounts of strontium, from 0.25, 0.50, and 0.75, to 1 mole using co-precipitation method. These synthesized nanoparticles were characterized by XRD, SEM, EDX, VSM, and FTIR for evaluating crystal structure, phase purity, morphology, composition, magnetic properties, and functional groups, respectively. Drug loading and drug release properties were determined using UV-vis spectroscopy, whereas MTT assay evaluated cytotoxicity.

Results

The findings confirmed the successful doping of iron oxide with strontium via XRD and EDX. SEM results confirmed spherical morphology for all and needle-like structure for 1 mole strontium doped sample. For VSM results, a single domain structure was established. It was also observed that the drug encapsulation efficiency increases with increased strontium content. Cytotoxicity results by MTT assay revealed increased cytotoxicity with increasing nanoparticle concentration, and ibuprofen-loaded nanoparticles showed higher cytotoxicity than un-loaded nanoparticles at the same concentration.

Conclusion

This study provided predominantly comparison of the cytotoxicity of ibuprofen-loaded and non-loaded nanoparticles on Hep-2 cancer cells at similar concentrations for the first time for both Fe₃O₄ particles and Sr-doped Fe₃O₄ nanoparticles and enclosed the impact of increasing Sr doping content on Fe₃O₄ nanoparticles.

Magnetic Iron oxide Nanoparticles

Ibuprofen

Drug Delivery

Cytotoxicity

Cell Viability

One of the most significant research areas in current science is nanotechnology, which allows researchers, scientists, and engineers to work at a molecular and cellular level. Nanoparticles are organic or inorganic materials, having at least one size dimension in a range of 1 to 100 nanometers (nm) (Samrot et al. 2021) and have enhanced properties compared to their bulk counterparts. Nanotechnology introduces various applications in the field of biomedical sciences. These applications include drug delivery, magnetic hyperthermia, bioimaging, and biosensors. Nanotechnology primarily focuses on therapeutic and diagnostic applications in the field of biomedical sciences (Davis et al. 1997) (D. K. Kim 2001) (https://en.wikipedia.org/wiki/Nanoparticle).

Applications of magnetic nanoparticles (MNPs) are growing faster nowadays. MNPs have properties like high surface area, high magnetization values, metal-enriched constituents, Chemical stability, ability to be surface functionalized, application at the molecular and cellular level, and their biocompatibility make them suitable for various applications in the field of biomedical sciences (Mattox et al. 2000). Their (synthesized nanoparticles) size and shape strongly depend upon synthesis conditions and surfactants, which play an essential role in synthesizing monodispersed nanoparticles (Rai-Choudhry 1997).

Significant research in nanotechnology in biomedical applications is regarding drug delivery. Mostly cancer is treated by chemotherapy which relies on the body's circulatory system to transfer anti-carcinogenic drugs to the desired area. It may damage healthy cells and organs during transportation, making this technique undesirable (Veiseh 2010). The introduction of targeted drug delivery can potentially replace harmful chemotherapy techniques. In this method, magnetic nanoparticles coated with the drug with the help of functionalization of a biocompatible layer help drug to be encapsulated to the surface. An external magnetic field concentrates the drug to the desired site where the tumor and cancerous cells are present. The drug is released with the help of a change in temperature, pH, or osmolality (Sethi et al. 2015). Superparamagnetic iron oxide nanoparticles (SPIONs) can be used as a drug-carrying agent upon a suitable synthesis process. SPIONs should be synthesized to prevent oxidation by the liver's reticuloendothelial system (RES). Due to their smaller size, these nanoparticles can easily cross biological blockades. Encapsulating SPIONs can increase their bioavailability and biocompatibility (Jeun et al. 2013).

Iron oxide nanoparticles have been studied extensively due to their inherent magnetic properties (showing superparamagnetic behavior), making them suitable for biomedical, electronic, and environmental applications. In addition to these properties, biocompatibility, stability, and environmentally friendly attributes make them ideal for biomedical applications. Among these magnetic nanoparticles, Magnetite (Fe₃O₄) and Hematite (Fe₂O₃) are the most widely used nanoparticulate system due to remarkable properties like superparamagnetic nature, biocompatibility, environmentally friendly, and stability. One of humankind's ancient iron ores is hematite as a pigment (Ren et al. 2016). Ferrites under the size of 100-nanometer act superparamagnetic and can be controlled using an external magnetic field (Mahmoudi et al. 2011).

The crystal structure of iron oxide nanoparticles contains oxygen anions and iron cations occupy tetrahedral and octahedral sites. In magnetite cubic, closed pack structure can be observed (Faraji et al. 2010) (Teja et al. 2009). Another ore is hematite, in which a packed hexagonal structure can be observed (Rafi et al. 2015). The properties of iron oxide nanoparticles depend upon the synthesized nanoparticle's size, shape, and crystallinity. These properties are dependent upon the synthesis route and reaction parameters. Iron oxide nanoparticles having a size below 20 nanometers (Mahmoudi et al. 2011) play an essential role in biomedical applications as these nanoparticles can quickly enter tumor sites crossing all barriers and creating therapeutic outcomes (Kandasamy et al. 2015). The shape and crystallinity also matter as round, and crystalline nanoparticles are preferred (Sun et al. 2006).

Magnetically guided iron oxide nanoparticles have high application in biomedical applications, especially in therapeutic applications like drug delivery. The external magnetic field is used to refer the nanoparticles to the tumor site and also aids in accumulating these nanoparticles at the tumor site (Wagstaff et al. 2012). The saturation magnetization of these nanoparticles plays an essential role in these applications, as the higher the saturation magnetization higher will be the control on the path of nanoparticles.

In previous research, iron oxide nanoparticles (IONPs) with cisplatin showed a 110-fold increase in cytotoxicity on cancer cell lines, improved drug loading, drug releases, and enhanced targeting of cancer cells (Hornung et al. 2015) (Nadeem et al. 2016). IONPs coated with Poly Vinyl Alcohol (PVA) and loaded with Dox showed reasonable control for guided delivery applications of drugs, and with human serum albumin (HSA) and lauric acid showed enhanced stability and a linear drug release pattern for approximately 72 hours (Zaloga et al. 2014) (Natesan et al. 2017). IONPs with chitosan showed enhanced encapsulation of artemisinin and higher drug accumulation at tumor sites. IONPs with polymerized paclitaxel showed higher magnetization values and enhanced anticancer activities (Jeon et al. 2016) (Silva et al. 2018).

Strontium acts as calcium and magnesium, so the human body differentiates between the two constructed on human body functions, which include renal uptake, mammary discharge, or gastrointestinal (stomach and intestine) absorption (Elumalai et al. 2020). In previous findings, strontium with Carboxymethyl Cellulose (CMC) showed potential in bioimaging applications. By adding strontium to biomaterials, more tenacity and durability are attained, and better control over degradation and enhanced drug release can be observed (Filippousi et al. 2015). Strontium hydroxyapatite nanorods with chitosan showed efficient transportation of hydrophobic drugs with no toxic effect (Jingzhe et al. 2012). Strontium with silica nanoparticles showed enhanced drug loading and release properties (Cuimiao et al. 2010). Ibuprofen-loaded mesoporous Strontium hydroxyapatite showed a regulated drug release profile (Farwa et al. 2008). For DNA interactive studies, increased binding efficiency was observed for Silver oxide nanoparticles doped with strontium (Wen-Yu et al. 2012). Strontium carbonate nanoparticles showed efficient release of anticancer drugs in an acidic environment around the tumor (Smith DM et al. 2013). Etoposide-loaded strontium carbonate nanoparticulate system showed enhanced anticancer potential (M Garbani et al. 2017). Porous spheres of strontium-doped hydroxyapatite were used as a matrix for transporting protein antigens showing an increase in the efficiency of allergen-specific immunotherapy (Khamsehashari et al. 2018) (Yurtdas et al. 2020).

Ibuprofen, a nonsteroidal anti-inflammatory drug (NSAID), is a derivative of isobutyl-phenyl-propionic acid used to treat pain from headaches, kidney stones, osteoarthritis, and dental and postoperative pain diseases. It is also effective against inflammatory conditions like arthritis. A previous study observed that a daily intake of 200 mg of ibuprofen reduces the risk of various types of cancer. Ibuprofen has more potent anticancer effects than other NSAIDs like aspirin, especially against breast cancer (Harris et al. 2005). Another study found that ibuprofen loaded on nanoparticles showed enhanced anticancer activity on the A-549 cancer cell line (lung carcinoma epithelial cells), which was 4–28 times higher than the free drug alone. It was observed that the antitumor activity of cisplatin was improved by adding ibuprofen (Endo et al. 2014). So, it was evident from the research that ibuprofen can be used as a potential therapeutic drug that aids in lowering the dose of cisplatin and reducing the challenges of toxicity and drug resistance.

This research essentially focused on synthesizing strontium-doped iron oxide nanoparticles by incorporating strontium in iron oxide in various concentrations (0, 0.2, 0.5, 0.75, and 1) moles via the co-precipitation method for drug delivery applications. Then ibuprofen was loaded on these nanoparticles, the drug loaded was determined on each sample, and the effect of strontium addition on drug loading and drug release was studied. The cytotoxic effect of these nanoparticles was also determined using an MTT assay, and the combined cytotoxicity result of both ibuprofen-loaded nanoparticles and bare nanoparticles were determined and compared. To the best of our knowledge, this combination of magnetite Fe₃O₄ with strontium nanoparticles and loading of ibuprofen with the specified ratio is studied for the first time for the cytotoxicity study of Hep-2 cancer cells.

2.1. Materials

Ferric chloride hexahydrate (FeCl₃.6H₂O (99% pure)) was purchased from Merck, ferrous chloride tetrahydrate (FeCl₂.4H₂O (99% pure)) was purchased from Sigma Aldrich, Ammonia Solution (32%) was used as a reducing agent, ibuprofen was used as a sample drug and Deionized water was used for the preparation of all solutions.

2.2. Synthesis of Fe₃O₄

Iron Oxide nanoparticles (magnetite Fe₃O₄) were synthesized by dissolving 0.2 moles of FeCl₃.6H₂O with 0.1 moles of FeCl₂.4H₂O in deionized water. The ratio was kept as 2:1 between Fe^+ 3 and Fe^+ 2 ions. The mixture was stirred for 1 hour; then ammonia was added quickly to the solution until the pH reached 11. The mixture was stirred at 800 rpm and 45°C temperature for 1 hour, and after that, the black magnetite nanoparticles were magnetically separated and washed several times with ethanol and deionized water. After that, the nanoparticles were dried at 60°C in the oven for 12 hours, and then the dried powder was further ground to obtain a fine powder, as seen in Fig. 1.

2.3. Synthesis of strontium-doped iron oxide nanoparticles

Strontium was added to synthesize strontium-doped iron Oxide nanoparticles to initial precursors and replaced by an iron atom (Fe^+ 2). Strontium was added in 0.25, 0.5, 0.75, and 1 mole of concentration with Fe^+ 3 and Fe^+ 2 ions. Synthesis parameters and procedure were the same as those used for synthesizing iron oxide nanoparticles via the co-precipitation route. An additional step of calcination at 700°C for 4 hours was performed to obtain the SrFe₂O₄ phase (Fig. 1). Eq. (1) was used to calculate molar ratios.

$$mass in \left(g\right)=\frac{Molarity \left(moles\right)\times Molar Mass\left(\frac{g}{mol}\right)\times Amount of Solvent \left(ml\right) }{1000 \left(\frac{ml}{l}\right)}$$

2.4. Drug loading on nanoparticles

The drug loading capacity of nanoparticles was determined on the calculation based on UV-Vis spectroscopy at 264 nm wavelength. Ibuprofen was used as a sample drug to load on these nanoparticles. Different dilutions were prepared using ethanol as a solvent containing 0.2 mg/ml to 1mg/ml ibuprofen solution in ethanol to determine the standard curve of ibuprofen. This linear plot of ibuprofen was obtained and further used to calculate the drug-loaded amount. For loading nanoparticles, 15 mg of nanoparticles were added to 10 ml of ethanol solution for each composition (Fig. 1). Then 15 mg of ibuprofen was added to the key to make the concentration of 1.5 mg of drug per ml (1.5 mg/ml). All samples were classified (Table 1) based on their composition, and each composition solution was stirred for 24 hours to load the drug. Then the drug-loaded sample was separated using a centrifugation process at 4000 rpm for 15 minutes. The remaining supernatant was analyzed using a UV-vis spectrophotometer absorption spectrum at 264 nanometers to check the amount of unloaded drug. Encapsulation efficiency was determined using Eq. 2.

$$Encapsulation Efficiency=\frac{Initial Drug Weight-Free Drug Weight in Supernatant}{Initial Drug Weight}$$

Table 1

Nanoparticles samples classification with different mentioned compositions
Sr. No.	Fe^+ 3 moles	Fe^+ 2 moles	Sr^+ 2 moles	FeCl₃.6H₂O grams	FeCl₂.4H₂O grams	SrCl₂.6H₂O grams
1	0.2	0.1	0	0.54	0.198	0
2	0.2	0.075	0.025	0.54	0.149	0.066
3	0.2	0.05	0.05	0.54	0.099	0.133
4	0.2	0.025	0.075	0.54	0.049	0.199
5	0.2	0	0.1	0.54	0	0.266

2.5. Drug release studies

The amount of drug released was determined by testing drug-loaded samples in a phosphate buffer saline solution. The drug-loaded samples were dispersed in phosphate buffer saline at 37°C under constant stirring. After selected time intervals, 3 ml of solution was withdrawn and refilled with fresh PBS, keeping the total volume the same. The amount of released drug was determined as a function of soaking time via UV-vis spectroscopy at 264 nm wavelength. The amount of drug released was evaluated based on Eq. 3.

$$Cumulative Drug Release\left(\text{%}\right)=\frac{Amount Of Drug Detected}{Amount Of Drug Loaded}\times 100$$

2.6. Cytotoxicity testing

MTT assay was used to determine the cytotoxicity of iron oxide, strontium-doped iron oxide, and ibuprofen-loaded nanoparticles.

2.6.1. Material and equipment

A Hep-2 cell laryngeal cancer cell line was obtained from the National Institute of Health (NIH). Iron oxide nanoparticles, strontium-doped nanoparticles, and ibuprofen-loaded nanoparticles were synthesized for characterization. The cell culture lab used a biosafety cabinet, an incubator with CO₂, 96 well plates, a single channel, and a multi-channel pipette. Other chemicals used included MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), dimethyl sulfoxide (DMSO), and Hank's Minimum Essential Medium Liquid (HMEM) which were procured in the required amount. BIOTEK absorbance microplate reader was used to take the absorbance values of each well. All these materials and equipment are displayed in Fig. 2.

2.6.2. MTT assay methodology

The Hep-2 laryngeal cancer cell line was obtained in 10 lac cells per milliliter (ml). To make it 10,000 cells per well, 10 microliters (µl) of cell solution was added to each well along with HMEM. These cells were incubated for 24 hours at 35ºC with CO₂ content at 5% CO₂. Iron oxide nanoparticles, Strontium doped Iron oxide nanoparticles, and Ibuprofen-loaded nanoparticles were prepared in different concentrations of 300 µg/ml, 400 µg/ml, and 500 µg/ml. The following concentration sample organized and their respective addition location in 96-well plates containing Hep-2 cancer cell lines are displayed in Fig. 3 and tabulated in Table 2.

Table 2

Differently prepared samples and their labeled symbols for 96-well plate
Sample	Concentration (µg/ml)	Symbol
Fe₃O₄	300	1.1
Fe₃O₄	400	1.2
Fe₃O₄	500	1.3
Sr_0.25Fe_2.75O₄	300	2.1
Sr_0.25Fe_2.75O₄	400	2.2
Sr_0.25Fe_2.75O₄	500	2.3
Sr_0.5Fe_2.75O₄	300	3.1
Sr_0.5Fe_2.75O₄	400	3.2
Sr_0.5Fe_2.75O₄	500	3.3
Sr_0.75Fe_2.25O₄	300	4.1
Sr_0.5Fe_2.25O₄	400	4.2
Sr_0.5Fe_2.25O₄	500	4.3
SrFe₂O₄	300	5.1
SrFe₂O₄	400	5.2
SrFe₂O₄	500	5.3
Ibuprofen loaded Fe₃O₄	300	B.1
Ibuprofen loaded Sr_0.25Fe_2.75O₄	300	B.2
Ibuprofen loaded Sr_0.5Fe_2.5O₄	300	B.3
Ibuprofen loaded Sr_0.75Fe_2.25O₄	300	B.4
Ibuprofen loaded SrFe₂O₄	300	B.5
Phosphate Buffer saline (Control)	Varied concentration	C

96-well plates with cells previously incubated for 24 hours were again incubated with nanoparticles at the same conditions for 24 hours. The total volume of each well was 200 µl. Phosphate buffer saline (PBS) was used as a control. MTT solution was prepared in phosphate buffer saline solution with a 5mg/ml ratio. After 24 hours of incubation, 20 µl of MTT was added to each well, and then the 96-well plate was incubated for 4 hours. After that, the excess solution was removed from each well, and 200 µl dimethyl sulfoxide (DMSO) was added to solubilize formazan crystals. Then absorbance reading was taken with the help of a BIOTEK absorbance microplate reader at a wavelength of 595 nm. The process is shown in Fig. 4.

To examine the cytotoxic effect of nanoparticles, MTT Assay was used. A Hep-2 cell line was used to investigate the cytotoxic effect of magnetite nanoparticles, strontium-doped iron oxide nanoparticles, and Ibuprofen-loaded nanoparticles. The Hep-2 cell line was treated with different concentrations (300, 400, and 500 µg/ml) of nanoparticles and was incubated for 24 hours to see the cytotoxic effect of these nanoparticles. The Cell viability was calculated by comparing the values of each cell with the absorbance values of the control. The formula used for calculating cell viability (%) is given by Eq. 4 below:

$$Cell Viability\left(\text{%}\right)=\frac{Absorbance \left(Sample well\right)}{Absorbance \left(Control well\right)}\times 100$$

3.1. XRD analysis

Patterns of Fe₃O₄ nanoparticles produced by Precipitation have been shown in Fig. 5. The peaks were observed at 2θ values of 30.04, 35.32, 42.80, 53.08, 57.015, and 62.66. These were found matched under standard JCPDS card number [01-088-0315] (Mahdavi et al. 2013), showing miller indices (220), (311), (400), (422), (511), and (440) at 2θ values of 30.2^°, 35.5^°, 43.2^°, 53.5^°, 57.2^°, and 62.7^° proving the formation of the inverse cubic spinal structure of Fe₃O₄. The results following the standard JCPDS card showed the development of black magnetite nanopowder. Sharper peaks indicated good crystalline structure had been formed. The broader peaks stated the formation of smaller crystallite sizes, calculated using the Debye–Scherer formula.

The remaining peak showed strontium-doped iron oxide nanoparticles in various molar ratios (0.25, 0.5, 0.75, and 1 mole). The results were in accordance to reference code [00-048-0156] (Berthet et al. 1992), showing orthorhombic structure showing diffraction angles of 2θ of 22.34^°, 24.16^°, 27.71^°, 33.08^°, 33.79^°, 35.51^°, 40.89^°, 42.92^°, 45.76^°, 49.38^°, 52.75^° and 57.62^° showing miller indices of (121), (041), (141), (002), (241) (311), (202), (400), (280), (331), (450), and (511) ( Singh et al. 2014) ( Al-Maswari et al. 2022). As the amount of strontium increases from (0.25 to 1) molar, the phase purity increases, and more peaks corresponding to the SrFe₂O₄ phase appear. At 1 molar peak at angles 2θ, 22.12, and 33.72 correspond to miller indices (121) and (241) occur. As the amount of strontium increased, a trend of growing crystallite size, as evident from calculated values through the Debye–Scherer formula, was observed as the strontium content increased peak with the highest intensity changed, which is clear from the graph. In a sample containing 0.75 and 1 moles of strontium, the highest intensity was at 2θ value 35.5^°, corresponding to the spinal structure, meaning a maximum number of atoms were lying in that plane. In contrast, samples containing 0.25 and 0.5 moles of strontium peak with the highest peak at 2θ value 32.89^° corresponded to the (002) plane. Meaning a maximum number of atoms are lying in that plane. Peak shift up to 1 degree can be observed due to strain-induced because of high-temperature sintering conditions. Calculated crystallite sizes are given in Table 3.

Table 3

The calculated crystallite size of synthesized nanoparticles
Sample	Crystallite Size (nm)
Sample	Mean	STDEV
Fe₃O₄	6.088	1.783
Sr_0.25Fe_2.75O₄	8.9541	4.6763
Sr_0.5Fe_2.5O₄	11.8	3.4498
Sr_0.75Fe_2.25O₄	10.048	4.4055
SrFe₂O₄	12.196	5.133

3.2. Morphological & elemental characteristics

Figure 6 shows synthesized nanoparticles on a glass substrate observed using a scanning electron microscope and EDS prepared by the co-precipitation method. The sample was prepared by dissolving a small number of nanoparticles in Deionized water, sonicating these nanoparticles for at least one hour, and then placing them on the glass substrate. Then gold plated it to make it conducive for further characterization. For Fe₃O₄, SEM images show the formation of nanoparticles in the nanometer range, with the majority of particles with spherical morphology. It can be observed from the image that smaller particles aggregated together to form larger particles due to dipole and magnetic interactions. Due to their smaller size and high specific surface area and surface energy, agglomeration can be seen. Clustering can be observed as no coating was used; these were bare magnetite particles. The particles show broad size distribution. The image shows the majority of nanoparticles below 100 nm, which is ideal for biomedical applications. Whereas for strontium doped Iron Oxide nanoparticles prepared by co-precipitation techniques sintered at 700°C, aggregation of particles can be seen due to the acceptable size of nanoparticles and due to which the surface energy increases, which leads to particles accumulation and formation of larger secondary particles as evident from the SEM images. The primary reason for the large aggregation was due to the combustion process (sintering particles at 700°C for 4 hours), which caused interfusion of nanoparticles through a chemical reaction. Most nanoparticles had spherical morphology, a size below 100 nanometers with moderated size distribution (Ghahfarokhi et al. 2020). It has also been observed that with strontium content up to 1 molar, more particles with wire shape morphology are seen, which is also detected in previous research (Shobegar et al. 2021).

Elemental analysis was done using energy dispersive spectroscopy (EDX) for the confirmation of doping of strontium to iron oxide nanoparticles. Figure 6 shows EDX results of SrFe₂O₄ prepared at 700°C establishing the presence of Strontium (Sr), Iron (Fe), and Oxygen (O) as significant elements present in the structure and their atomic ratios following the given literature of SrFe₂O₄. Impurities peaks detected were of gold due to the gold plating of the sample to make it conductive. A secondary silicon peak was observed as the sample was placed on a glass slide due to a small quantity of silicon detected. Carbon content was also seen because the powder was placed on carbon tape to attach to the sample holder for characterization. It can be observed from the results that strontium content increases from 7.7 to 39.7 atomic weight % with the increase in the doping ratio of strontium from 0.25 to 1 mole (Yusoff et al. 2017).

3.3. Functional group analysis

Figure 7 shows FTIR spectra of Ibuprofen-loaded Fe₃O_4, Sr_0.25Fe_2.75O_4, and SrFe₂O₄. The Fe₃O₄ peak at 544 cm^− 1 can be seen, associated with Fe-O bond stretching in Fe₃O₄ (Mousavi et al. 2019). The peak at 1640 cm^− 1 is associated with a carboxylic group related to the presence of ibuprofen on the surface. Absorption bands (6) and (7) between 900 cm^− 1 to 1100 cm^− 1 were related to bending vibrations associated with O-H bonds. SrFe₂O₄ (A) and Sr_0.25Fe_2.75O₄ (B) bands between 600 cm^− 1 to 400 cm^− 1 (10) (11) corresponded to the formation of tetrahedral and octahedral sites confirming the presence of metal-oxygen stretching band in ferrites—a weak band at 435.12cm^− 1 (11) associates with the octahedral Fe^+ 3-O^− 2 group complex. Bands at 542.16 cm^− 1 (A) and 561 cm^− 1 (B) correspond to the Fe^+ 3/Sr^+ 2-O group (Campos et al. 2020). Bands at 1641 cm^− 1 (1), 1550–1560 cm^− 1 (2), 1221–1234 cm^− 1 (5), and 770–900 cm^− 1 (8)(9) were footprints of the presence of ibuprofen in samples corresponding to the carboxylic group, νC = C, νC-C, and δCH2 groups (Khamsehashari et al. 2018). These four bands can be identified in all three spectrums, showing the presence and loading of ibuprofen molecules.

3.4. Magnetic properties

Figure 8(a) shows the hysteresis loops of Fe₃O₄ nanoparticles, and strontium doped (0.25, 0.5, and 0.75 molar concentration) Iron oxide nanoparticles, from which magnetic behavior of synthesized nanoparticles can be obtained. Magnetic properties like magnetic coercivity (H_c), saturation magnetization (M_s), isotropic property or squareness ratio (M_r/M_s), and remanence magnetization (M_r) from the hysteresis loops are obtained. It can be observed that with the increase in strontium doping content decrease in saturation magnetization (M_s) can be observed. An increase in magnetic coercivity (H_c) and remanence magnetization (M_r) can be observed, associated with a shift in magnetic property from soft to hard magnetic material. With the increase in strontium content, it becomes harder to magnetize and de-magnetize the material hence enhanced coercivity is observed. The trend observed in previous research is that up to a specific size range (crystallite size) of nanoparticles, coercivity increases with an increase in the size of nanoparticles. Hence similar results are observed in our case. A growth in remanence magnetization is attributed to increased exchange interaction with increased strontium content. The squareness ratio of our samples ranges from 0.07 to 0.35, which is less than 0.5, confirming the formation of a single domain structure. The superparamagnetic behavior of synthesized Fe₃O₄ nanoparticles can be documented as the squareness ratio is near zero. Magnetic parameters of synthesized nanoparticles can be observed in Fig. 8(b, c, d, e) and Table 4.

Table 4

The measured magnetic parameters of prepared nanoparticles with different compositions
Material	M_s (emu/g)	M_r (emu/g)	H_c (Oe)	M_r/M_s	K
Fe₃O₄	34.04	2.25	43.04	0.07	1526.40
Sr_0.25Fe_2.75O₄	28.19	6.43	187.46	0.23	5504.68
Sr_0.5Fe_2.5O₄	22.86	7.39	446.58	0.32	10633.72
Sr_0.75Fe_2.25O₄	14.94	5.20	725.87	0.35	11298.62

3.5. Drug loading and drug release characteristics

3.5.1. Standard curve of ibuprofen

Different dilutions were prepared to find the standard curve of ibuprofen. The graph was plotted between absorbance and concentration 0.2mg/ml, 0.3mg/ml, 0.5mg/ml, and 0.9 mg/ml solutions were prepared in ethanol to find the standard curve of ibuprofen. Absorbance was measured at 264 nm to detect the amount of drug in absorbance at different dilutions. Figure 9(a) and Table 5 give mean absorbance values and standard ibuprofen curve.

Table 5

Absorbance values of ibuprofen-loaded samples with different concentrations
Concentration	Absorbance		Mean
mg/ml	1	2	Mean
0.2	0.4125	0.352	0.38225
0.3	0.5825	0.5423	0.5624
0.5	0.9399	0.879	0.90945
0.9	1.5139	1.66	1.58695

3.5.2. Drug loading studies

Iron oxide nanoparticles and strontium doped nanoparticles with strontium content 0.25, 0.5, 0.75, and 1 molar were loaded with ibuprofen. The samples were prepared by loading 15 mg of the drug in 10 ml ethanol to make a 1.5mg/ml solution. The solution was stirred for 24 hours and then centrifuged. The amount of drug remaining in the solution was detected at 264 nm wavelength us a UV-vis spectrophotometer which was then compared to a standard curve of ibuprofen to calculate the amount of drug relative to that absorbance value. The absorbance value of samples at 264 nanometers determined by UV-vis spectrophotometer is shown in Fig. 9(a).

Ibuprofen at 1.5 mg/ml has an absorbance value of 2.6201, calculated from a standard curve of ibuprofen shown above. The remaining drug content, detected at 264 nm from a UV-vis spectrophotometer, was measured by the standard curve of ibuprofen. The relative drug weight was calculated against absorbance values. The encapsulation efficiencies of all nanoparticles are given in Table 6. Fe₃O₄ nanoparticles showed an encapsulation efficiency of 34.31%, whereas 0.25 moles strontium doped iron oxide nanoparticles showed an encapsulation efficiency of 44.01%, which is calculated to be the highest among all sample's encapsulation efficiencies. The Increased drug loading can be attributed to the fact that strontium is an alkaline earth metal and creates more basic sites on nanoparticles, enhancing bonding with the carboxylic group in ibuprofen. Other samples with strontium content of 0.5 and 0.75 moles showed encapsulation efficiency of 35.96% and 36.33%, which was better than the encapsulation efficiency of Fe₃O₄ alone, as seen in Fig. 9(b). The encapsulation efficiency of 0.5 and 0.75 moles doped iron oxide nanoparticles is less than 0.25 moles doped Iron oxide nanoparticles, which could be attributed to particle size increases with the increase in strontium content hence reducing specific surface area hence reducing the content of the drug-loaded (Vallet-Regi et al. 2021). In previous studies, it has been observed that drug loading depends on many factors, including textural properties like pore volume and pore size.

Table 6

The investigated encapsulation efficiency (%) of pure Fe₃O₄ and strontium-doped iron oxide nanoparticles loaded with ibuprofen
Sample	Absorbance Value	Relative Drug Weight (mg/ml)	Initial Drug Weight (mg/ml)	Encapsulation Efficiency (%)
Fe₃O₄	1.7266	0.985309	1.5	34.3
Sr_0.25Fe_2.75O₄	1.475	0.839791	1.5	44.01
Sr_0.5Fe_2.5O₄	1.684	0.960670	1.5	35.96
Sr_0.75Fe_2.25O₄	1.6744	0.955118	1.5	36.33
SrFe₂O₄	1.8527	1.058241	1.5	29.45

The smaller the pore size, the more minor the release rate. The higher the pore volume, the higher the amount of drug encapsulated. Secondly, the specific surface area is vital in the drug-loading phenomenon. It has been observed that the higher the specific surface area, the greater the amount of drug loaded. Functionalization also enhances drug-loading capabilities. In our case, our samples were non-porous, and there was no coating or functionalization element. Therefore specific surface area plays a significant role in determining the drug loading capabilities of these nanoparticulate samples (Tabia et al. 2019) (Doadrio et al. 2014) (Norouzi et al. 2020). An increase in particle size can be observed from SEM results, and crystallite size calculated from XRD results with the help of the Scherer equation. From SEM results, it can be observed that with the increase in strontium content, an increase in overall particle size can be monitored, and a shift from spherical to rod shape morphology can be seen, which is larger hence having a low specific surface area. It is also observed that with increased strontium content, crystallite size increases from 6nm to 13 nm, showing an increase in particle size.

3.5.3. Drug release studies

In vitro drug release profile was studied for all drug-loaded samples performed for 48 h in pH 7.4 phosphate buffer held at 37°C. The cumulative Concentration (CC) of the drug released was calculated with the help of UV-vis absorption spectra at 264 nm, shown in Table 7. It can be observed that for sample Sr_0.25Fe_2.75O₄ maximum amount of drug (0.58 mg/ml) was released regarding all other samples, which is evident from the previous results Sr_0.25Fe_2.75O₄ showed the maximum encapsulation efficiency among all samples. Sr_0.75Fe_2.25O₄ showed the maximum release percentage of the drug of 97.5% concerning the amount of drug loaded on these samples within 48 hrs. Fe₃O₄ showed a minimum percentage of drug release of about 63.15% within 48 hours.

Table 7

Cumulative and percentage drug release data of drug-loaded nanoparticles with different compositions of Sr-doped Fe₃O₄
Time ((Hrs.)	Fe₃O₄		Sr_0.25Fe_2.75O₄		Sr_0.5Fe_2.5O₄		Sr_0.75Fe_2.25O₄		SrFe₂O₄
Time ((Hrs.)	CC (mg/ml)	% Released	CC (mg/ml)	% Released	CC (mg/ml)	% Released	CC (mg/ml)	% Released	CC (mg/ml)	% Released
3	0.095142	18.48129	0.276403	41.87917	0.148641	27.5567	0.238924	43.9199	0.111047	25.06135
6	0.128282	24.91885	0.348253	52.76566	0.192435	35.67574	0.301093	55.348	0.152134	34.33405
12	0.161735	31.41708	0.423939	64.23314	0.239335	44.37057	0.350561	64.44136	0.208855	47.13492
224	0.238028	46.23694	0.494002	74.84884	0.293366	54.38749	0.418907	77.00494	0.276125	62.31662
336	0.293916	57.09315	0.54963	83.27725	0.350642	65.00593	0.480052	88.24486	0.341625	77.0989
448	0.325147	63.15996	0.580428	87.94364	0.394757	73.18448	0.530717	97.5583	0.399526	90.16604

Figure 10(a, b) shows the ibuprofen release profile of all samples. In the first 6 hours, we can see a burst release of the drug for all samples. For Sr_0.75Fe_2.25O₄ and Sr_0.25Fe_2.75O₄, 55.3% and 52.76% of the loaded drug were released, and after that, for 42 hours, a steady amount of drug release was observed for all samples. Graph A shows the cumulative drug release percentage for every sample calculated based on the amount of drug loaded on each nanoparticulate sample. It can be observed that during the first 3 hours, the burst release of the drug occurred from all samples. Graph B shows the drug release percentage of these samples, which is the ratio of the drug released to the amount of drug loaded on these samples. Graph B in Fig. 10 shows the cumulative amount of drug released by all samples for 48 hours. It can be observed that Sr_0.25Fe_2.75O₄ showed the highest amount of drug released. The release profile of all samples showed better performance for the Fe₃O₄ drug release profile.

3.5.4. Cytotoxicity analysis

For 10,000 cells per well, Cell viability was calculated. Results in Table 8 showed an increasing trend in cytotoxic effect as the concentration of the particles increases for all samples, which agrees with previous research (Norouzi et al. 2020) (Kanagesan et al. 2013). Significant cell viability reduction can be seen at higher concentrations for magnetite and strontium-doped Iron oxide nanoparticles. It can be seen from the results that the cell viability of SrFe₂O₄ nanoparticles at 300 µg/ml concentration shows maximum cell viability of 112.4%, and ibuprofen-loaded Sr_0.5Fe_2.5O₄ showed minimum cell viability of 52.9% and maximum cytotoxic effect. As the concentration of nanoparticles increases, a cytotoxic effect can be seen, and a decrease in % cell viability can be observed for both magnetite and strontium-doped iron oxide nanoparticles.

Table 8

Percentage cell viability (%) of different concentrations of nanoparticles and ibuprofen-loaded nanoparticles
Material	Concentration (µg/ml)
Material	Cell Viability %	300	400	500	300 Ibuprofen Loaded
Fe₃O₄		105.7	70.3	54.1	60.8
Sr_0.25Fe_2.75O₄		86.7	89.8	69.9	54.4
Sr_0.5Fe_2.5O₄		109.	84.2	69.6	52.9
Sr_0.75Fe_2.25O₄		108.1	91.6	83.5	74.5
SrFe₂O₄		112.4	89.5	61.9	81.6

Figure 11(a) compares the cytotoxicity of all samples at 300, 400, and 500 µg/ml concentration. At 300 µg/ml, all SrFe₂O₄ showed maximum cell viability of 112.4%, whereas Sr_0.25Fe_2.75O₄ showed minimum cell viability of 86.7%. At 400 µg/ml, all Sr_0.75Fe_2.25O₄ showed maximum cell viability of 91.6%, whereas Fe₃O₄ showed minimum cell viability of 70.3%. At 500 µg/ml, all Sr_0.75Fe_2.25O₄ showed maximum cell viability of 83.5%, whereas Fe₃O₄ showed minimum cell viability of 54.1%. So, from the following results, it can be observed that by doping strontium to Iron oxide nanoparticles, a reduction of cytotoxic effect can be seen at all concentrations, which is due to the biocompatible nature of strontium.

Figure 11(b) compares the cytotoxic effect of nanoparticles and ibuprofen-loaded nanoparticles at 300 µg/ml concentration. It can be observed that ibuprofen Loaded nanoparticles have a higher cytotoxic effect than non-loaded nanoparticles at the same concentration. For each sample, it can be seen that ibuprofen-loaded nanoparticles show higher cytotoxicity. Ibuprofen-loaded Sr_0.75Fe_2.25O₄ nanoparticles showed a maximum cytotoxic effect and minimum cell viability of 52.9%. So, it can be observed from the results that ibuprofen-loaded nanoparticles are effective against the Hep-2 cancer cell line.

In this study, iron oxide (Fe₃O₄) and strontium doped (0.25, 0.5, and 0.75 to 1 mole) nanoparticles were developed via the co-precipitation method, and ibuprofen was loaded on them. XRD, EDS, and FTIR results confirmed the formation of strontium-doped iron oxide nanoparticles by showing relevant peaks and confirming different percentages of strontium doped to iron oxide nanoparticles. Nanoparticles possessed crystallite sizes ranging from 6–13 nanometers, and most synthesized nanoparticles were spherical, as observed by SEM. VSM results confirmed a single domain structure. FTIR results confirmed the successful loading of ibuprofen. Sr_0.25Fe_2.75O₄ showed maximum encapsulation efficiency of 44.01%. Results showed that increased strontium concentration from 0.25 to 1 molar decreased encapsulation efficiency due to a reduction in the specific surface area that occurred with increased strontium content. Overall, strontium-doped iron oxide nanoparticles showed better drug-loading capabilities than iron oxide nanoparticles alone. The results of in vitro drug release of Sr_0.25Fe_2.75O₄ showed the maximum amount of drug released in 48 hours (0.58 mg/ml), liberating 87% of its loaded drug, which was better compared to other nanoparticulate samples. Cytotoxicity (MTT) results exposed increased cytotoxicity with the increasing concentration of nanoparticles. It was detected that strontium doping reduces the overall cytotoxicity of samples for every concentration. It was also witnessed that ibuprofen-loaded nanoparticles were more cytotoxic to Hep-2 cancer cells than un-loaded ones. Sr_0.25Fe_2.75O₄ and Sr_0.5Fe_2.5O₄, loaded with ibuprofen, showed a maximum cytotoxic effect, reducing cell viability to 54.2% and 52.9% of Hep-2 cancer cell line at 300 µg/ml concentration. Results proved that strontium-doped iron oxide nanoparticles up to 0.25 molar concentration improved drug loading capabilities, better drug release profile, and higher cytotoxic effects. So, strontium doped up to 0.25 molar to iron oxide nanoparticles enhances the properties of iron oxide nanoparticles for biomedical applications, especially in drug delivery applications.

Author contributions

All authors contributed to the drafting and scientific revision of the manuscript.

Funding

This research received no specific grant from public, commercial, or not-for-profit funding agencies.

Competing Interests

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript.

Data availability statement

All data generated or analyzed during this study are included in this published article.

Conflict of interest statement

All the authors have no conflict of interest

Ethical Approval

Not applicable

Acknowledgments

This research was conducted at the School of Chemical and Materials Engineering (SCME), National University of Sciences and Technology (NUST), Sector H-12, Islamabad, Pakistan. The author acknowledges the financial and administrative support from the department.

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Novel Cytotoxicity Study of Strontium (Sr) Doped Iron Oxide (Fe₃O₄) Nanoparticles Aided with Ibuprofen for Drug Delivery Applications

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1. Introduction

2. Material and Methods

2.1. Materials

2.2. Synthesis of Fe₃O₄

2.3. Synthesis of strontium-doped iron oxide nanoparticles

2.4. Drug loading on nanoparticles

2.5. Drug release studies

2.6. Cytotoxicity testing

2.6.1. Material and equipment

2.6.2. MTT assay methodology

3. Results and Discussion

3.1. XRD analysis

3.2. Morphological & elemental characteristics

3.3. Functional group analysis

3.4. Magnetic properties

3.5. Drug loading and drug release characteristics

3.5.1. Standard curve of ibuprofen

3.5.2. Drug loading studies

3.5.3. Drug release studies

3.5.4. Cytotoxicity analysis

4. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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Novel Cytotoxicity Study of Strontium (Sr) Doped Iron Oxide (Fe3O4) Nanoparticles Aided with Ibuprofen for Drug Delivery Applications

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Abstract

Figures

1. Introduction

2. Material and Methods

2.1. Materials

2.2. Synthesis of Fe3O4

2.3. Synthesis of strontium-doped iron oxide nanoparticles

2.4. Drug loading on nanoparticles

2.5. Drug release studies

2.6. Cytotoxicity testing

2.6.1. Material and equipment

2.6.2. MTT assay methodology

3. Results and Discussion

3.1. XRD analysis

3.2. Morphological & elemental characteristics

3.3. Functional group analysis

3.4. Magnetic properties

3.5. Drug loading and drug release characteristics

3.5.1. Standard curve of ibuprofen

3.5.2. Drug loading studies

3.5.3. Drug release studies

3.5.4. Cytotoxicity analysis

4. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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Novel Cytotoxicity Study of Strontium (Sr) Doped Iron Oxide (Fe₃O₄) Nanoparticles Aided with Ibuprofen for Drug Delivery Applications

2.2. Synthesis of Fe₃O₄