Green synthesis, characterization and drug-loaded iron oxide nanoparticles derived from Nerium oleander flower extract as a nanocarrier for in vitro antibacterial efficacy

Application of drug conjugated iron oxide hematite (α-Fe2O3) nanoparticles are of tremendous interest in biomedicine nowadays. Meanwhile, green production of iron oxide nanoparticles is gaining favour due to its sustainability, ease of usage, and biocompatibility. Therefore, this work reports on the use of hexahydrate ferric chloride and nerium oleander flower extract to synthesize nanoscaled hematite (α-Fe2O3) iron oxide particles conjugated with various drugs for antibacterial agents. Diverse morphological, physicochemical, structural, optical, and magnetic characteristics have been characterized using FESEM, EDX, XRD, UV–vis, FTIR, Raman and vibrating sample magnetometer. The synthesis of the polyshaped iron oxide nanoparticles, with average sizes ranging from 47.2 ± 20 nm, was accomplished. Furthermore, temperature-dependent variations in magnetic behavior were observed during calcination. The XRD and Raman spectra revealed hematite (α-Fe2O3) type formation of iron oxide nanoparticles. Only calcinated IO-NPs at high temperatures (700 °C) demonstrated low coercivity and residual magnetism, which revealed weak ferromagnetic ordering; other calcinated samples, including nascent ones, showed incredibly weak ferromagnetic ordering. Besides, the effectiveness of drug-encapsulated iron oxide nanoparticles against bacteria in vitro was examined. It was interesting to observe that gentamycin-coated IO-NPs tended to be more susceptible to S. aureus than E. coli bacteria, but streptomycin-conjugated IO-NPs showed the reverse trend. However, as compared to the nascent sample and the high temperature (700 °C) calcinated sample, both antibiotic-loaded IO-NPs displayed better inhibitory abilities.


Introduction
One of the most significant advances in medicine to date, the discovery of antibiotics in 1928 enabled the treatment of formerly deadly illnesses, saving millions of lives [1].Microbial pathogen infections are a constantly rising concern with serious global ramifications, affecting millions of people every day and being a leading cause of mortality in both adults and children [2,3].Antibiotics significantly reduced infection-related mortality, but misuse of them gave rise to the problem of antimicrobial resistance [4][5][6].In order to avoid the negative effects of antimicrobial resistance, current initiatives emphasize the use of alternative antimicrobial agents and substances.It is anticipated that antibiotic resistance alone would cause 10 million deaths each year by 2050, and that early detection and treatment will effectively reduce the mortality rate caused by bacterial infections [7].However, the growing problem of antibiotic resistance, which is particularly prevalent in healthcare applications continues to be a challenge for academics and medical practitioners alike and poses a danger to the fundamental principles upon which modern medicine was founded.Given the gravity of the situation, it is critical to find effective approaches for the early diagnosis, prevention, and treatment of these illnesses.Until recently, several attempts based on nanoparticles have been done to detect and eliminate harmful germs [8][9][10].
In this context, iron oxide nanoparticles (IO-NPs) are a viable alternative for developing drug-delivery systems with controlled drug release [11][12][13][14][15][16].Bacterial illness diagnosis and treatment are critical for bacterial infection prevention and control.Understanding the role of magnetic nanoparticles in bacterial diagnosis and treatment might help guide the development of magnetic nanoparticles-based materials for the detection and treatment of bacterial diseases.Iron oxide NPs are one of the most well-known metals and metal oxide NPs.The remarkable quality of iron oxide nanoparticles has broadened the spectrum and applications of these materials in medical, therapies, and a variety of other sectors, including gas sensors, electrochemical, magnetic, and energy storage [17,18].IO-NPs are often employed in imaging, drug delivery, biosensors, and other applications due to their superparamagnetic characteristics.Furthermore, because of their special traits, which include biocompatibility, high magnetic properties, low toxicity, and catalytic activity, they have emerged as viable candidates for a variety of biological applications [19][20][21].
The use of nanoparticles for drug delivery offers several benefits over traditional approaches.Iron oxide nanoparticles in a nanodrug delivery system have a competitive advantage over other nanomaterials due to unique properties such as strong magnetic properties and a larger surface to volume ratio [22][23][24].Additionally, it helps to achieve site-specific medication delivery, which helps to resolve issues with appropriate bioavailability and promotes the elimination of tumor cells [25].It has been demonstrated that leveraging the magnetic and biological characteristics of iron oxide nanocarriers to load or bind drugs is a successful strategy for increasing the therapeutic efficacy of these treatments [26,27].Besides, many drugs have unfavourable properties that can be removed by conjugating to iron oxide nanoparticles, including limited solubility, high toxicity, nonspecific binding, and very short circulation half-lives [28][29][30][31].Furthermore, these nanocarriers are nontoxic, biocompatible, biodegradable, and efficiently removed from the body via iron metabolism [32][33][34].
On the other hand, the technique of synthesis of IO-NPs is critical since it regulates their size, shape, surface characteristics, and applications.The particle size, morphology, and magnetic properties of iron oxide nanoparticles often influence their biomedical applications.Iron oxides can be found in a variety of polymorphs, the most common of which are crystalline α-Fe 2 O 3 (hematite), γ-Fe 2 O 3 , β-Fe 2 O 3 (maghemite), and γ-Fe 2 O 3 , Fe 3 O 4 (magnetite) [35,36].Amorphous forms are also common at high pressure.Iron oxide NPs have been synthesized utilizing chemical and mechanochemical techniques, such as sol-gel synthesis, template-assisted synthesis, reverse micelle synthesis, hydrothermal synthesis, co-precipitation, laser ablation, arc discharge etc. [20,37].By merely modifying the precursor iron salts, various kinds of iron oxides, such as nanorods, porous spheres, nanohusks, nanocubes, distorted cubes, and self-oriented flowers, may be produced using relatively similar synthetic processes.These innovative methods are simple to use, affordable, and manage shape in an environmentally friendly way.Iron oxide surface modification is essential in addition to the synthesis in order to achieve better functionalization, molecular conjugation, and biocompatibility.
Natural resources, on the other hand, such as plant, floral, and food extracts, are gaining attention and support in the synthesis of IO-NPs due to their capacity to function as stabilizing and reducing agents throughout the synthesis process.Moreover, the biological methodology for producing iron oxide nanoparticles using plant extract has several benefits over both physical and chemical techniques, including being easier, more cost effective, and beneficial to the environment.Additionally, the presence of diverse phytochemicals including flavonoids, alkaloids, terpenoids, and other hydroxyl-containing functional groups on the surface of the NPs prevents agglomeration and makes it easier to produce NPs with uniform particle size [38,39].
In this study, we report on the synthesis of iron oxide nanoparticles utilizing iron tetrachloride hexahydrate as a precursor and different phytochemicals contained in Nerium oleander flower extract as reducing and capping agents.Nerium oleander flower extract usually contains various phytochemicals such as alkaloids, flavonoids, terpenoids, cardiac glycosides, saponins, tannins, carbohydrates, minerals, and other hydroxylcontaining functional groups [40].The presence of these diverse phytochemicals aids in the reduction process during the formation of iron oxide nanoparticles via green synthesis.Additionally, the hydroxyl-containing functional groups on the surface of the NPs prevent agglomeration and make it simpler to regulate the homogeneity of the particle sizes.A wide range of physicochemical, morphological, structural, surface functional, optical, and magnetic properties have been studied of the synthesized IO-NPs.For in vitro antibacterial activity, the nanoformulated drugs streptomycin and gentamycin conjugated iron oxide nanoparticles were further studied.

Experimental section 2.1. Materials
Analytical research grade ferric chloride hexahydrate (FeCl 3 , 6H 2 O) and sodium hydroxide (NaOH) were purchased from Loba Chemie (India) and were used without further purification.Whatman filter paper (grade 1), acetone, ethanol, and isopropyl alcohol were all purchased from local chemical vendors.Double distilled water (18.2MΩ.cm) was used all throughout the experiments unless otherwise specified.

Green synthesis of iron oxide nanoparticles
Nerium oleander (family: Apocynaceae), a subtropical to tropical shrub with five-petaled funnel-shaped blooms in clusters that are generally purple, pink, or white, was initially collected from a neighbouring university campus (GPS locations: 30°25' N, 77°04'E).The flowers were thoroughly cleansed with tap water to remove dust particles.The flowers were washed and sundried before being crushed into a fine powder.A 1000 ppm flower extract solution was then prepared.In brief, 1.00092 g of nerium oleander flower powder was dissolved in 1000 mL of DI water while stirring with a magnetic stirrer.The heat is then increased to 80 °C and maintained at that temperature for 30 min.After cooling down, the flower extract was filtered twice with Whatman no.1 filter paper (11 μm, medium flow filter paper).The pH of the extract solution was noted to be around 5.0.The IO NPs were synthesized using ferric chloride hexahydrate (FeCl 3 , 6H 2 O) as iron precursor.In order to create a homogenous 0.1 M ferric chloride solution, ferric chloride was first dissolved in 550 mL of deionized water and constantly stirred at room temperature for 15-20 min.The same amount of Nerium oleander flower extract solution was added to ferric chloride solution and stirred continuously with a magnetic stirrer at ambient temperature.
This time, the pH of the solution mixture was acidic (pH: ∼3).Following that, 1 M NaOH was added dropwise to the solution mixture heated to 60 °C until the pH became basic (∼pH: 10).The stirring was kept going at a rate of 600 rpm to finish the reaction until the formation of an intense brown coloured solution was obtained thereby confirming the formation of iron oxide nanoparticles.It was then kept running for a few hours to stabilize.The precipitate was removed from the solution by centrifuging it for 5 min at 7000 rpm.The resulting IO-NPs were then rinsed many times with distilled water before being dried in a hot air oven overnight at 60 °C and ground into a fine powder with a pestle and mortar.The NPs were calcinated for three hours in a high temperature tube furnace at three distinct temperatures: 300, 500, and 700 °C.The dried powder of IO NPs was used for further study and physical characterization.The entire synthesis process is illustrated in figure 1.

Characterizations and instrumentations
Using a REMI CPR-30 Plus centrifuge, the sample was ultra-centrifuged.A Shimadzu UV-2600 spectrophotometer with a 1 nm step in the wavelength ranges 200-800 nm was used to acquire UV-vis absorption spectra.A REMI MS-500 magnetic stirrer was used to mix the mixture.The kind of surface functionalization was investigated using a Shimadzu IR Spirit Fourier transform infrared (FTIR) spectrophotometer in the wavelength range 4000-400 cm −1 .The crystalline nature of the NPs was verified using a Bruker D8 Advance diffractometer set to 40 kV, 40 mA, and a non-monochromatic Cu K α x-ray with an angular range of 5-90°and an angular step of 0.02°.A Zeiss field-enhanced secondary electron microscope (FESEM) set to 20 kV was used to analyse the morphologies of synthesized NPs.Energy-dispersive x-ray spectroscopy (EDX) was used to evaluate the composition of IO-NPs.Magnetization was evaluated at room temperature using a vibrating sample magnetometer (VSM-7404, Lake Shore).

Bacterial culture
Microbial assays were utilized to compare the suppression of sensitive bacteria growth generated by known quantities of the test antibiotic and a control drug.E. coli and S. aureus, two commonly used gram-negative and gram-positive bacteria, were grown in nutritional broth overnight at 37 °C for 24 h.A spectrophotometer was used to quantify the optical density at 620 nm and analyse bacterial growth curves [41].

Preparation of antibiotic coated iron oxide nanoparticles
The use of nanomedicine for drug distribution may improve antibiotic effectiveness for therapy.Nanosystems for prescribing antibiotics and infection site targeting have several benefits over traditional formulations.In this study, streptomycin and gentamycin antibiotic-coated IO-NPs were prepared to investigate the role of nanoparticles in microbial activity.In the beginning, 10-30 μL of freshly synthesized IO-NPs (either nascent or calcinated @ 700 °C sample) were mixed in order to examine the cumulative impact of each standard antibiotic.

Microbial assay
The in vitro antibacterial activity of streptomycin and gentamycin-coated IO-NPs was investigated using the well diffusion technique on Mueller-Hinton agar plates.To begin, Mueller-Hinton agar plates were infected with spore suspensions (20 μL) of the microorganisms under investigation.The antibiotic-coated IO-NPs were then put on agar plates and incubated for at least an hour at 25 °C to allow for pre-incubation diffusion, which reduced the effect of timing variations when various solutions were utilized.The plates were examined for antibacterial activity after 12-24 h of incubation at 37 °C by measuring the breadth of the inhibition zones for each bacterial culture [41].

Assessment of antimicrobial activity
Antibacterial activity was measured by increasing fold area and given as the diameter of the zone of inhibition.Experiments were carried out in triplicate, and the average inhibitory zone diameter of pure antibiotic and antibiotic coated-IO-NPs, as well as the standard deviation, were calculated [41].

Results and discussion
3.1.Morphological, compositional, structural and physicochemical characteristics of Nerium oleander derived IO-NPs The morphology of the synthesised IO-NPs as assessed by FESEM is presented at different magnifying scales in figures 2(a)-(c).The FESEM image clearly shows that the synthesised iron oxide nanoparticles are not homogenous in nature and, in some cases, agglomerate.
The particles are polygonal in form, with a size distribution of 47.2 ± 20 nm as shown in figure 2(d).The enormous agglomerated clusters formed possibly due to the absence of a capping layer or the accumulation of microscopic reducing agent building blocks.Similar experimental work on the bio-green synthesis of Iron oxide NPs from leaves, fruits, seeds, seed coat, flowers, peels, petals, or whole plant has been published in the literature, as shown in table S1 in the supplemental document.In addition, magnetic nanoparticles have a tendency to clump together and create enormous clusters due to the high surface area to volume ratio, strong dipole-dipole interactions, van der Waals, attractive forces, and magnetic forces, which results in an increase in particle size.The elemental composition of the IO-NPs was also determined via EDX analysis.The EDX analysis shown in figure 3 clearly demonstrates the existence of corresponding L α at 0.73 keV, and another K α line at 0.52 keV due to the presence of Fe and O atoms, respectively in the nanoparticle.
According to the table in the inset of figure 3, the fraction of iron and oxygen atoms in the irradiated area is 37.01 at.% and 51.50 at.%, respectively.The presence of zinc (Zn) in trace concentrations (0.11 at.%) is most likely owing to minerals found in nerium oleander flower extract.It is crucial to keep in mind that the carbon that can be observed in EDX spectra (C K α at 0.2 keV) may come from interactions with organic molecules during preparation or with air.Any organic contamination has a propensity to create hydrocarbon, the concentration of which may increase throughout the course of the experiment, on the sample surface beneath the electron beam.However, the presence of Au at around 2.1 keV owing to M α is due to gold coating of the NPs.Hence, it can be concluded from EDX analysis that IO-NPs were successfully synthesized using nerium oleander in a green synthesis process.
The structural properties of nerium oleander derived IO-NPs was characterized by XRD. Figure 4(a) illustrates a comparative XRD patterns of as-synthesized (denoted as nascent) and calcinated (300, 500 and 700 °C) IONP samples.
X-ray diffraction reveals that high temperature calcinated samples (either 500 or 700 °C) developed polycrystalline iron oxide nanoparticles with strong peaks from the (012), ( 104), ( 110), ( 113), (024), ( 116 [42].Meanwhile, hematite is the most stable form among others.Various synthesis processes, including as chemical co-precipitation, hydrothermal, sol-gel synthesis, and others, have reported the production of IO-NPs in hematite phases [43].Often the particle size and shape may be easily adjusted by changing numerous factors related to their production techniques.Therefore, on the basis of EDX and XRD studies it is confirmed that the hematite (α-Fe 2 O 3 ) phase has been synthesized successfully.
Moreover, the average crystallite size (d np ) was calculated using the conspicuous peaks from Debye-Scherrer's formula [25]: Cos where λ = 0.15406 nm is the wavelength of CuK α radiation, β is the full width at half maxima (FWHM) of the diffraction peak in radians and θ is the Bragg's diffraction angle, κ is a constant.The average crystallite size was estimated to be around 21.8 nm.Additionally, the Williamson-Hall (W-H) plot and the following relationship were used to calculate the lattice strain from the FWHM of the diffraction peaks [42]: b q e q = + kl Cos 4 Sin , where ε denotes the effective strain.Plotting the term (βcosθ) with respect to (4sinθ) for the preferred orientation peaks of IO-NPs reveals the crystallite size and related strain, as well as the y-intercept and slope of the fitted line, respectively [44,45].The W-H plots for nascent and calcinated IO-NPs as shown in figure 4(b), demonstrate that the strain is exceedingly small.On the other hand, the various surface functional groups presence in the shell of IO-NPs were investigated by FTIR. Figure 5 depicts the FTIR spectra of as-prepared and calcined Fe 2 O 3 -NPs.The existence of distinct reducing agent functional groups connected with IO-NPs was established by FTIR analysis (400-4000 cm −1 ) of all samples (nascent and calcinated samples).
The spectra of Fe 2 O 3 -NPs exhibit a distinct band at 570 cm −1 , which is associated with iron oxide's Fe-O vibrations [46].The peak of the signal at 3750.57cm −1 represents the -OH bond stretching from the aqueous phase.The existence of C=C and N=C=N bond stretching vibrations is represented by the peak between 1979.4 and 2401.7 cm −1 .Furthermore, the peaks at 1351.4 cm −1 are caused by C-H stretching vibration [47,48].To further clarify the formation of hematite (α-Fe 2 O 3 ) phase, Raman spectra of nascent and calcinated samples were investigated.Figure 6 shows the comparison of Raman spectra of nascent and calcinated (500 and 700 °C) IO-NPs samples.
The Raman spectra recorded up to 700 cm −1 contains four phonon lines associated with transverse optical (TO) vibrational modes, two A 1g modes, and two E g modes.Peaks around 221 and 396 cm −1 belong to A 1g modes, whereas the other peaks at 281 and 602 cm −1 correspond to E g modes.Aside from TO-mode lines, longitudinal (LO) mode lines are also observed at 1295 cm −1 .The positions and intensities of these peaks are consistent with previously reported results for hematite (α-Fe

Optical characteristics of nerium oleander derived iron oxide nanoparticles
Diffuse reflectance spectroscopy (DRS) in UV-vis is a scientific technique to measure the optical band gap energy of nanomaterials, particularly thin films or powders, that is more effective than typical absorbance or transmittance modes [51].Here in this study, DRS is utilized to evaluate the optical band gap of IO-NPs as shown in figure 7(a).Schuster-Kubelka-Munk (SKM) reemission function has been used in this regards: ) where R ∞ is the reflectance.
Then the bandgap, E g can be easily obtained extrapolating the straight-line plot of the optical spectral reflectance n n according to Tauc equation [52]: ) where h denotes Planck's constant, v denotes vibration frequency, A denotes constant, and E g denotes bandgap.The exponent n varies depending on the type of transition, with n = 2 or 3 for direct transitions and n = 1/2 or 3/2 for indirect transitions.Figure 7(b) depicts the Tauc plot of nascent and calcined IO-NPs samples.The indirect band gap falls marginally in calcinated materials (300 °C-700 °C) compared to nascent samples (1.548 eV).

Magnetization measurement of nerium oleander derived iron oxide nanoparticles
The magnetic characteristics of the synthesized IO-NPs were assessed by measuring the hysteresis loops at room temperature (300 K) with a vibrating sample magnetometer equipped with a Lake Shore model 7404 and a maximum applied field of 1 T. Figure 8 depicts magnetization curves (M-H loops) for nascent and calcinated magnetic IO-NPs measured at room temperature.
It is worth mentioning that the magnetization did not achieve saturation at the highest applied magnetic field of 10 kOe, and no notable hysteresis loops were found either for nascent or calcined samples (300 or 500 °C) with no noticeable remanent magnetization and almost null coercivity fields (see inset of figure 8 for details).This behavior revealed that the IO-NPs were paramagnetic at room temperature.However, a distinctive hysteresis loop without magnetic saturation observed in 700 °C calcinated IO-NPs demonstrated their week ferromagnetic ordering.Similar observation of week ferromagnetism of hematite (α-Fe 2 O 3 ) nanoparticles above Morin transition temperature has also been observed earlier [53].
It is worthy to note that nanosized hematite particles often display a variety of unusual magnetic behaviours as compare to bulk hematite.It has been reported earlier that the Morin temperature (∼263 K for bulk) of α-Fe 2 O 3 nanoparticles can be reduced by decreasing particle size, which even tends to disappear for particle size below 10 nm [54,55].Hematite behaves as an antiferromagnetic material when the spins order antiparallel below the Morin temperature, while weak ferromagnetism is manifested above Morin temperature due to ordering of spins [56,57].Meanwhile, superparamagnetic behavior and spin-glass properties have been reported for IO-NPs size below 10 nm [58,59].The observed hysteretic activity indicates that the sample is weakly magnetic at room temperature.Only the 700 °C calcinated sample shows a substantial hysteresis loop and no magnetization saturation.According to previous research, hysteretic behavior is caused by surface spin disorder, whereas the linear component of the magnetization curve indicates the contribution of the particle's antiferromagnetic core [60].Although the relationship between particle shape and physical characteristics of nanosized materials has been extensively researched in the literature, it remains unclear how particle shape and magnetic properties are associated in magnetic materials and requires further study [56,57,59].
The values of residual, saturation magnetization, and coercivity increase when the calcinated temperature rises from 300 to 700 °C, as can be shown in table 1.In light of the aforementioned discussion, it is anticipated that the weak ferromagnetic behavior of a sample that has been calcined at 700 °C may be caused by changes in nanoparticle size and shape anisotropy with calcination temperature.

Bacterial growth inhibition assay and drug delivery applications of antibiotic conjugated-IO-NPs
The in vitro antibacterial activity of drug-conjugated IO-NPs was initially examined using an agar diffusion assay to compare the growth inhibitory effects of two commonly used representative bacterial species, one Grampositive bacteria, S. aureus and the other Gram-negative bacteria, E. coli at various time intervals ranging from 1 to 24 h.
Figures 9(a)-(b) shows the digital photographs of antibacterial study of streptomycin drug (10 μL) loaded iron oxide nanoparticles in nascent condition.According to figure 9(c), streptomycin-encapsulated IO-NPs significantly outperformed streptomycin or IO-NPs alone in terms of antibacterial activity.This is because the compound is essentially made up of a group of antimicrobial agents that typically inhibit protein synthesis and a close-knit group of aminoglycosides.The mechanisms of killing bacteria by streptomycin-encapsulated IO-NPs are the disintegration of bacterial wall and the subsequent leakage of cytoplasmic contents and inactivation of proteins responsible for DNA and RNA replication.The increase in fold area for streptomycin-encapsulated IO-NPs has clearly demonstrated the same.
The basic mechanism of action of these medicines is the suppression of protein synthesis or genetic translation.When iron and streptomycin are mixed, phospholipids are formed [61].The sulfate group forms a covalent bond, enveloping the nanoparticles in a coating [62].Following that, the conjugate mostly leads to cell  annihilation via two mechanisms: at first by limiting protein synthesis, and secondly, by damaging cell membranes.It thereby produces a highly efficient drug carrier [63].The bactericidal activity of IO-NPs, in contrast, is triggered on by reactive oxygen species (ROS) produced by hydroxyl radicals, superoxide radicals, singlet oxygen, and hydrogen peroxide.ROS can damage the proteins and DNA of bacteria, which results in oxidative stress and bactericidal activity in IONPs [64,65].Despite this, the generation of free radicals during the oxidation-reduction process continues to be the driving force behind the induction of oxidative stress on bacterial cells, leaving them non-viable.The greater the interaction, the more ROS produced, culminating in the dissolution of bacterium membranes [66].
Antimicrobial drugs are often capable of releasing active ionic species via interaction with active sites (-SH) of proteins in bacterial cells and causing cell lysis [64].However, it is interesting to note that streptomycinconjugated IO-NPs (nascent) demonstrated more resistant against E. coli bacteria than S. aureus.
Similar to this, IO-NPs coated with gentamycin were also studied.The antibacterial activity of three distinct samples is depicted in figures 10 The antibacterial efficacy was also examined with 700 °C calcinated IO-NPs.Figures 11 and 12 represent the relevant studies using streptomycin and gentamycin-conjugated IO-NPs, respectively.
As with the nascent one, calcinated IO-NPs demonstrated comparable antibacterial effectiveness trends for both drug loadings.
As compare to nascent one, the zone of inhibition for streptomycin coated IO-NPs (calcined @ 700 °C) was nearly the same against Gram negative bacteria (E.coli), whereas it was somewhat larger against Gram positive bacteria (S. aureus), as shown in figures 11(c) and 12(c), respectively.Therefore, based on the above studies, it is obvious that streptomycin and gentamycin drug coated iron oxide nanoparticles were highly efficient against both Gram positive and Gram negative bacteria, as evidenced by a larger fold increase in the zone of inhibition diameter.Similar results have been reported in the literature when iron oxide nanoparticles were produced by co-precipitation employing different plant or fruit extracts as reducing agents [67][68][69][70][71][72][73].Besides, in-vitro studies against several gram positive and gram negative pathogens, including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis, revealed high antibacterial efficacy of the Gentamicin coated IO-NPs conjugation have also been reported by Bhattacharya et al [74].
Nevertheless, the iron oxide nanoparticles had no negative or adverse effects on microbial activity.As a consequence, the effectiveness of Fe 2 O 3 -NPs as a drug carrier may be examined further for drug delivery systems.Although the mechanism of contact between nanoparticles and the constituents of microorganisms' outer membrane is still unknown, interactions between the nanoparticles and the outer membrane's building blocks are likely to produce structural alterations or degeneration.The mechanism(s) of putative improvement of antibacterial activity of iron oxide nanoparticle conjugates, in our opinion, remains an open subject that requires more investigation.
These results confirmed that the drug-encapsulated IO-NPs had potent effects on both types of pathogenic bacteria, thereby killing them either through member disruption after penetration into the bacterial cells or by rupturing the inner membrane.Another study reported that metal NPs disrupt the stability of lipopolysaccharides present in the outer cell membrane of the bacterial cell, thus increasing the permeability of the outer membrane and the peptidoglycan layer of the cell wall, which might have been recognized and captured by the antibiotics, followed by the action of the NPs conjugated with antibiotics on the pathogen [ 75,76].Hence, we presume the same situation in the present case, wherein the streptomycin/chloramphenicol -encapsulated IO-NPs could have affected the lipid layer of the outer cell wall, leading to degradation of the outer membrane, thereby causing destruction of the bacterial pathogen.According to another hypothesis, bioactive compounds of the Nerium oleander flower extract serve as capping and stabilizing agents in the synthesis of the Iron Oxide NPs together with the antibiotics; could have affected the cell wall surface, thereby rupturing the cell membrane and lysing the vital cell organelles, exposing them to the extracellular environment and ultimately leading to the death of the bacterial cells [77].
Based on the foregoing, it is reasonable to believe that IO-NPs encapsulated with streptomycin and gentamycin antibiotics will become an efficient drug carrier against bacterial infections due to increased surface area, chemical stability, and appropriate size of the generated NPs.However, substantial animal studies are required before employing IO-NPs as possible antibacterial agents.

Conclusions
In summary, nanoscaled iron oxide particles were synthesized in ambient conditions using an inexpensive ecofriendly green synthesis technique that utilized ferric chloride hexahydrate as a precursor and various phytochemicals from nerium oleander flower extract as reducing and capping agents.The morphological, structural, physicochemical, optical, and magnetic characteristics of synthesized iron oxide nanoparticles were investigated using FESEM, EDX, XRD, FTIR, Raman, UV-Visible, and VSM.FESEM micrographs reveal that polyshaped iron oxide nanoparticles with an average size distribution in the range of 47.2 ± 20 nm were produced.In accordance with XRD examination, IO-NPs that have been calcined at low temperatures (300 °C) or nascent sample are not well crystalline, but samples that have been calcined at higher temperatures (500 and 700 °C) are well polycrystalline with hematite (α-Fe 2 O 3 ) phase.A vibrating sample magnetometer was also used to study the magnetic properties at room temperature.The magnetization failed to reach saturation even when a maximum applied magnetic field of 10 kOe was used, and neither nascent nor calcined samples (300 or 500 °C) showed any sign of coercivity (H c ) or residual magnetism (M r ).The IO-NPs were shown to be extremely week ferromagnetic at room temperature by this result.However, a substantial M r (0.234) value and hysterics loop without magnetic saturation observed at 700 °C calcined IO-NPs demonstrated its week ferromagnetic ordering.The variance in M r and H c reveals that magnetic characteristics vary with calcination temperature and are mostly controlled by extrinsic features such as crystallite size and phase of the nanoparticles.In order to study the effectiveness of in vitro antibacterial therapy, drug nanofomulation was also carried out.For this purpose, streptomycin and gentamycin-encapsulated IO-NPs were synthesized as nanoformulated antibiotics for in vitro antibacterial activity on Mueller-Hinton agar plates against Gram-positive (S. aureus) and Gram-negative (E.coli) bacteria.It was interesting to figure out that streptomycin-conjugated IO-NPs were more resistant to E. coli bacteria than S. aureus, although gentamycin-coated IO-NPs displayed the opposite trend.However, as compared to the nascent one, the high temperature (700 °C) calcinated sample, both antibiotic-loaded IO-NPs displayed better inhibitory capacities.The findings imply that iron oxide nanoparticles conjugated with antibiotic drugs might be useful in treating wound infections caused by S. aureus and E. coli.These findings support the promising potential of these nanoparticles and give compelling evidence for developing this material as an effective therapeutic alternative for treating various infections.

Figure 1 .
Figure 1.Schematic entire green synthesis of iron oxide nanoparticle from nerium oleander flower extract.

Figure 3 .
Figure 3. Compositional analysis by EDX spectra of nerium oleander flower extract derived iron oxide nanoparticles.

Figure 5 .
Figure 5. FTIR spectrum of iron oxide nanoparticles derived from nerium oleander flower extract.

Figure 7 .
Figure 7. (a) UV-vis diffused reflectance spectra of iron oxide nanoparticles synthesized from nerium oleander flower extract.(b) Tauc plot to determine the optical band gap.The influence of calcination temperature are also shown.

Figure 8 .
Figure 8. M-H loops of nascent and calcinated (300, 500 and 700 °C) iron oxide nanoparticles synthesized from nerium oleander flower extract.Inset shows the magnified view of M-H loop.
(a)-(b): Gentamycin antibiotic alone, IO-NPs alone, and gentamycin conjugated IO-NPs.Similar antibacterial activity was demonstrated, as shown in figure 10(c), with gentamycin coated IO-NPs (nascent) achieving the largest zone of inhibition against S. aureus when compared to E. coli.

Figure 9 .
Figure 9. (a)-(b) Digital photographs of in vitro antibacterial activity of the streptomycin drug (10 μL) loaded iron oxide nanoparticles against Gram-negative (E.coli) and Gram-positive (S. aureus) bacteria in Mueller-Hinton agar plates.(c) Zone of inhibition observed for nascent IO-NPs alone, streptomycin antibiotic alone and streptomycin-conjugated IO-NPs (nascent).

Figure 11 .
Figure 11.(a)-(b) Digital images showing the antibacterial activity of iron oxide nanoparticles (calcinated @ 700 °C) loaded with the antibiotic streptomycin (10 μL) against S. aureus and E. coli bacteria on Mueller-Hinton agar plates.(c) Corresponding plot of zone of inhibition.

Figure 12 .
Figure 12. (a)-(b) Digital photographs of the antibacterial activity of iron oxide nanoparticles (calcinated @ 700 °C) loaded with the antibiotic gentamycin (10 μL) against the microorganisms S. aureus and E. coli on Mueller-Hinton agar plates.(c) Comparative studies on zone of inhibition.

Table 1 .
Magnetic properties (M s .M r and H c ) of nascent and calcinated nerium oleander derived IO-NPs.