Antimicrobial and antioxidant therapy with bioactive plant molecules on Fe₃O₄ phytohybrid nanoplatforms

Abstract

Background

Nanobiomedicines have gained increasing attention for their potential to improve efficacy and are emerging as a promising therapeutic paradigm. Magnetic nanoconjugates loaded with bioactive drugs have the advantage of sustained circulation in the bloodstream and significantly reduced toxicity of therapeutic agents in a precise manner. The well-developed surface chemistry of Fe₃O₄ has led to the development better tools, promoting them as nanoplatforms with potential technological applications in biomedical sciences.

Results

Fe₃O₄ phytohybrids with Laxmitaru extract as the primary coating and loaded with Eugenol and Ylang-Ylang essential oils were successfully synthesized. The X-ray diffraction technique has revealed the high purity nanoparticle materials, as no additional impurity peaks were observed. Fourier transform infra-red spectra have confirmed the presence of a primary coating of Laxmitaru extract and a secondary layer of essential oil, as additional peaks and broadening are observed in drug-loaded Fe₃O₄ nanoparticles. Magnetic susceptibility values indicate the material's superparamagnetic nature. Transmission electron microscopy images have ensured that the particles were spherical, monodispersed, and in the range of 4.30 nm to 13.98 nm. Antimicrobial studies show inhibition zones on the microorganisms S. Aureus and E. Coli with enhanced activity. Drug entrapment efficiency studies revealed the encapsulation of drug molecules onto Fe₃O₄-Laxmitaru composite. Dynamic light scattering studies confirm the increase in hydrodynamic size, indicating the loading of essential oils and the decrease in polydispersity index ensures monodispersed nanoparticles. The antioxidant study showed the essential oils retained their antioxidant activity even after they were conjugated on Fe₃O₄-Lax composites.

Conclusions

Laxmitaru phytochemical-coated Fe₃O₄ nanoparticles were successfully conjugated with Eugenol and Ylang-Ylang essential oils. Our results provide a model therapeutic approach for the development of new alternative strategies for enhancing antimicrobial and antioxidant therapy, with potential advantages in the field of nanobiomedicine.

Background

Nanoscience and nanotechnology are the most important research areas in modern science and allow researchers to produce important advances in healthcare science. Nanoscale drug delivery platforms have gained importance over the past few decades, which have shown promising clinical results in treating varieties of cancer and inflammatory disorders. This selective administration enhances therapeutic drug efficacy at targeted sites while minimizing the adverse side effects. The use of nanomaterials as carriers for drugs or other bioactive therapeutic agents has been widely investigated, with the focus to improve therapeutic effect, on-site release, and lowering of side effects of the administered drugs. Among magnetic materials, Fe₃O₄ nanoparticles (NPs) are extensively studied and are considered as promising drug carriers in nanomedicine, owing to their various advantages such as their unique size, excellent biocompatibility, strong affinity, extremely low toxicity, biodegradability, surface reactivity, superparamagnetic nature and other properties making them preferable from traditionally used materials [1,2,3,4,5]. Many regular treatment therapies have limitations in efficacy, which has led to the necessity of nanomedical innovations. A balanced perception of the relationship between inorganic nanomaterials and the biological system has led to the emergence of better solutions for individualized therapy regimes. The composite inorganic–organic NPs have significantly enhanced the identification, quantification of specific disease biomarkers, improving the clinical translation and utility of nanomaterials in the field of medicine [6, 7]. Several metals and metal oxide NPs have been investigated for biomedical use. ZnO NPs synthesized through eco-friendly and natural sources has shown promising results as antibacterial, antibiofilm and other biomedical applications [8].

Antimicrobial approach based on magnetic nanoparticles (MNPs)

Microbial infections are listed among the major health concerns, occurring globally, where in the recent past a variety of fungi, yeasts, and several other pathogenic microorganisms, are responsible for developing a drug resistance thereby enhancing frequency and occurrence of infections. Antimicrobial resistance (AMR) has led to high rates of mortality, economic losses, worsening of health care, increased diagnosis and treatment losses, thus giving rise to a need to develop new antibacterial agents and strategies against drug-resistant super-bugs [9]. One of the most enticing antimicrobial strategies is based on the use of MNPs to transport and control the release of active drugs at the diseased site. Due to the development of AMR to synthetic drugs, a strategy to use plant compounds having antimicrobial properties is being harnessed. MNPs being permeable to tissues and cells can be magnetically targeted to reach specific sites inside the body. This way the bioactive drugs can be delivered at the diseased site which conventional drugs may not reach by themselves. Additionally, this could also minimize undesirable side effects [10]. Due to targeted action, the toxic effects of drugs on healthy tissues can be avoided.

Fe₃O₄ conjugates as promising nanosystems

MNPs are attractive materials to develop novel routes for Targeted Drug Delivery (TDD) because of their well-developed surface chemistry, significant surface/area ratio, and superparamagnetism. Moreover, applications in biosystems require MNPs to be stable in water at pH 7 and in the physiological environment [10]. Fe₃O₄ NPs are preferred as it contains Fe⁺² ions that have the potential to act as an electron donor. Though, they are magnetic materials, their residual magnetization is zero, a property observed to be beneficial to avoid coagulation which further lowers the in vivo agglomeration. Additionally, their high surface activity is vulnerable to oxidation in the air, affecting their magnetic and dispersibility property. Therefore, the functionalization of Fe₃O₄ NPs with hydrophilic and biocompatible polymers coating, prevents oxidation and provides colloidal stability, enhanced dispersibility, and ensures chemical binding/conjugation sites for drug molecules and other therapeutic agents to the superparamagnetic Fe₃O₄, thereby optimizing the biomedical utility for TDD [11,12,13,14].

The drug can be thus targeted to the desired site using an external magnetic field, avoiding delivery to healthy tissues. Fe₃O₄ NPs have been used with various synthetic and natural drugs to inhibit microbes that could cause serious infectious diseases. As they occur naturally in the human heart, spleen, and liver; they have been approved for clinical use by Food and Drug Administration (FDA) [3, 10]. Being nanosized, smaller concentrations of drug can be adsorbed on the particles and thus reducing the side effects and the exposure of the drug concentration at the infection site. The release of the drug can be controlled with respect to time of release and dosage [10, 15]. Bifunctional Fe₃O₄@Ag NPs prepared by reducing Ag ions on the surface of Fe₃O₄ NPs show both superparamagnetic and antibacterial properties [16]. Fatty acids assisted biologically synthesized superparamagnetic γ- Fe₂O₃ NPs ensure the stability to the NPs. They were investigated for antimicrobial, antibiofilm, and anti-cancer biomedical applications. Magnetic property was an advantage to boost the biological activity of γ- Fe₂O₃ NPs by the application of external magnetic field or in blend with other therapeutic drugs [17].

Simarouba glauca commonly known as Laxmitaru (Lax) plant was used as herbal medicine against dysentery. The crude bark and leaf water extracts contains active phytochemicals having several pharmacological properties such as antipyretic, hemostatic, anticancerous, antiparasitic, and anthelmintic besides antidysenteric properties [18]. Quassinoids are the major group of phytochemicals present and to date approximately 200 of them are isolated and structures elucidated [19]. In the present study, the concentrated water extract of Lax was explored as the natural biocompatible organic coating on Fe₃O₄ NPs to stabilize the molecule. Thus making it immune friendly, being non-toxic with known medicinal benefits. Quassinoids present in the plant extract have antimalarial and cytotoxicity effects against several human cancer cell lines [18].

Eugenol (Eug) is an allyl chain-substituted guaiacol, i.e., 2-methoxy-4-(2-propenyl) phenol. It is a member of the allylbenzene class, colorless to pale yellow oily liquid extracted from certain essential oils especially from clove oil, nutmeg, cinnamon, and bay leaf. It is slightly soluble in water and soluble in organic solvents. Eug is used in perfumeries, flavorings, and in medicine as a local analgesic (topical), antiseptic, antifungal, and an anesthetic. Eug possesses significant antioxidant, anti-inflammatory, and cardiovascular properties [20].

Ylang-Ylang (Yla) is an essential oil from Cananga Odorata, a perfumed tree (medicinal) bearing yellow flowers. Extensively used by perfume, food industry, and aromatherapy for its powerful floral fragrance and flavor. It is water-insoluble and contains sesquiterpenes, monoterpenes, phenols, methyl benzoate, benzyl acetate, geranyl acetate, linalool, geraniol which contribute to its medicinal property. It has a vast spectrum of pharmacological activities such as antioxidant, antibiofilm, antimicrobial, antifungal, antiinflammatory, insect repellent, antidiabetic, antimalarial, antiseptic, additionally it has curative properties for internal infections of the colon, etc.[21].

The objectives of this investigation were to synthesize Lax-coated Fe₃O₄ phytochemical nanoparticles, load them with plant-derived bioactive drugs Eug and Yla, to characterize and confirm their structural morphology, the percentage of drug loading, evaluate antimicrobial and antioxidant activity. These characteristics contribute to the description as promising molecules in the development of a new strategy of drug delivery.

Methods

All materials used were of Analytical Reagent grade as procured without any further purification. Deionized and nitrogen-purged water was used to prepare all the solutions. Reagents used were FeCl₃.6H₂O (99% pure), FeSO₄.7H₂O (98.5% pure), NaOH (Laboratory Reagent), Eugenol (Laboratory Reagent) 98%, Ylang-Ylang 98% were used as commercially procured. Laxmitaru (dry leaves) were obtained from the local market.

Synthesis of Fe₃O₄ NPs

FeCl₃.6H₂O and FeSO₄.7H₂O were taken in the ratio of 2:1 and dissolved in acidified deionized water. Then 2 M NaOH was added dropwise, with constant stirring and the reaction temperature was maintained at 60 °C. The pH was maintained above 11. The black precipitate of Fe₃O₄ obtained was sonicated for 1 h, repeatedly washed with deionized water and ethanol, and finally separated and dried. The net reaction is: 2Fe⁺³ + Fe⁺² + 4OH⁻ → Fe₃O₄ + 4H⁺

In-situ synthesis of pristine Fe₃O₄ and Fe₃O₄—Lax-coated NPs

Lax solution was prepared by boiling Lax dry leaves in (approx. 100 mL) pure water for 1 h The content was filtered and used as Lax extract. To this, 2 M NaOH (100 mL) was added. Stoichiometrically prepared ferrite solution (100 mL) in the ratio of 1:2 (FeSO₄.7H₂O and FeCl₃.6H₂O) was added dropwise to the Lax-extract. The suspension was incubated for 1 h at 90 °C with gentle stirring as reported elsewhere [22]. The reaction pH was maintained above 11 by adding 2 M NaOH solution. The Fe₃O₄-Lax colloidal suspension was washed with de-ionized water and then with ethanol. The phytohybrid-coated Fe₃O₄-Lax NPs were dried at 60 °C and then further treated for loading.

Biogenic synthesis of Fe₃O₄-Lax-Eug and Fe₃O₄-Lax-Yla nanoconjugates using essential oils

The Fe₃O₄-Lax NPs were then functionalized with bioactive plant-derived drugs Eug and Yla to prepare the double-layered Core/shell/secondary shell nanomaterials. Eug solution (20 mg/mL in ethanol) and Yla solution (1 mL/5 mL in ethanol) were prepared and added to a known quantity of Fe₃O₄-Lax NPs. The suspensions were stirred magnetically for several hours, allowed to settle overnight, separated and dried [14]. The drugs were attached to Fe₃O₄-Lax by adsorption.

Materials characterization

X-ray powder diffraction (XRD) was used to determine the crystal structure & particle size in the 2Ɵ range of 20°–80° (Philips PW 1840 XRD diffractometer). Fourier transformed infra-red (FTIR) (Thermo Nicolet iS5 spectrometer) spectra were recorded in the wave range of 4400 to 400 cm⁻¹. Magnetic measurements was studied using vibrating sample magnetometer (VSM) (Lake Shore Model 7404S) and saturation magnetization (Ms) values were calculated. The morphology of the nanoconjugate materials was analyzed by high-resolution transmission electron microscopy (HRTEM) (JOEL JEM 2100F instrument). The size distribution of the synthesized Fe₃O₄ NPs before and after surface modification was measured using dynamic light scattering (DLS) (Bechman Coulter Delsa Nano Analyzer) at scattering angle of 90° and 25 °C as discussed elsewhere [15]. The average diameters were calculated from the three individual measurements.

Bioactivity evaluation of Fe₃O₄—Lax phytohybrids loaded with Yla and Eug drug

Antimicrobial studies were carried out by the zone of inhibition (ZI) method using the borewell agar diffusion technique discussed elsewhere [15, 23,24,25]. This method was used to quantitatively determine the level of inhibition of the bacterial strains produced by the bioactive plant drugs Eug and Yla loaded on Fe₃O₄-Lax NPs. Antibacterial activity was tested against bacterial reference strains Staphylococcus Aureus (S. Aureus) (ATCC 6538) and Escherichia Coli (E. Coli). The nanoconjugates were individually dispersed in alkaline buffer and dimethylsulfoxide (DMSO) to obtain the test solutions of concentration 2 mg/mL and 40 μg/mL, respectively. Subsequently, wells were bored into Mueller Hinton agar plates, using a sterile 10-mm-diameter cork borer and aseptically dispensed with 100 μL of test solutions. The temperature and time duration of incubation was maintained at 37 °C for 24 h, respectively. The plates were monitored for ZI which were recorded in mm. For consistency, the experiment was performed in triplicate.

Drug entrapment efficiency (% EE) studies were carried out to for the quantitative estimation of the amount of drug Eug and Yla loading capacity of Fe₃O₄-Lax phytohybrids using a UV–Vis spectrophotometer (Thermo Scientific Evolution 201) in the range of 200 to 800 nm as reported elsewhere [11]. A solution of a known quantity of Eug and Yla was prepared in ethanol. To this, a known quantity of Fe₃O₄-Lax was added. The dispersion was stirred for 45 min, allowed to stand for 10 min, and then coated compounds were separated. Spectra of the solution which contained the unadsorbed Eug and Yla was recorded before and after the adsorption process.

In vitro free radical scavenging activity (RSA) was performed by the following the method given elsewhere [11, 26, 27]. In brief, the decolorization property of the stable DPPH (2,2 diphenyl-1-picrylhydrazyl) was used in presence of synthesized Fe₃O₄- phytohybrids. A measured quantity of synthesized Fe₃O₄ NPs was added to the known molarity of DPPH solution and incubated in dark for 15 min at room temperature. The intensity of purple color decreased significantly with time to yellow–brown and was measured using UV–Vis spectrophotometer (Thermo Scientific Evolution 201 model) at λ_max of 514 nm. The percentage RSA of Fe₃O₄ NPs- Lax and Eug was determined using the following expression.

RSA (%) = [(Ac -As)/Ac] × 100, where Ac is the absorbance of the control DPPH at zero min; As is the absorbance in the presence of the Fe₃O₄ nanocomposite sample after 15 min.

Results

Structural morphology and particle size

X-ray powder diffraction (XRD) peaks were analyzed and indexed using Joint Committee on Powder Diffraction Standards files and compared with magnetite standards. Figure 1 shows the XRD pattern for all the Fe₃O₄ conjugates. Crystallite size measurements were determined from the full width at half maxima (FWHM) of the strongest reflection of (311) peak using the Debye–Sherrer approximation formula, which assumes the small crystallite size to be the cause of line broadening. The crystallite sizes calculated are given in Table 1.

Fig. 1

XRD patterns of a Fe₃O₄, b Fe₃O₄-Lax, c Fe₃O₄-Lax-Eug, and d Fe₃O₄-Lax-Yla

Table 1 Particle size, Saturation magnetization (Ms) and Scavenging activity of phytohybrid Fe₃O₄ nanoconjugates

Fourier transformed infra-red (FTIR) spectroscopy

FTIR was recorded_, in the % transmittance mode. Figure 2 shows the FTIR spectra of Fe₃O₄, Fe₃O₄-Lax, Fe₃O₄-Lax-Eug & Fe₃O₄-Lax-Yla. The absorption peak frequencies for all the peak intensities are shown in Table 2.

Fig. 2

FTIR spectra of a Fe₃O₄, b Fe₃O₄-Lax, c Fe₃O₄-Lax-Eug and d Fe₃O₄-Lax-Yla

Table 2 Assignments of the absorption bands in the FTIR spectra (cm⁻¹)

Magnetic measurements

Vibrating sample magnetometer (VSM) and saturation magnetization (Ms) values. Superparamagnetism plays a key role in targeting carriers in biomedical applications and the lack of hysteresis is one criterion to identify the product as superparamagnetic. The magnetic characterization was done at room temperature, with a magnetic field in the range of − 15,000 Oe to + 15,000 Oe. The parameters obtained from the hysteresis loops were Ms, remanence magnetization (Mr) and coercivity (Hc). Figure 3 shows the VSM spectra of Fe₃O₄, Fe₃O₄-Lax, Fe₃O₄-Lax-Eug, Fe₃O₄-Lax-Yla. The Ms values are shown in Table 3.

Fig. 3

VSM graphs of a Fe₃O₄, b Fe₃O₄-Lax, c Fe₃O₄-Lax-Eug and d Fe₃O₄-Lax-Yla

Table 3 Zone of inhibition for (a) references/controls and (b) Fe₃O₄-Lax nanoconjugates

High-resolution transmission electron microscopy (HRTEM)

Surface morphology and particle size. Figure 4 shows the HRTEM images of pure Fe₃O₄ NPs and coated conjugates. All the images illustrate that the average particle size was less than 20 nm. HRTEM analysis revealed that the mean core size of (a) Fe₃O₄ NPs was 4.30–12.78 nm, (b) Fe₃O₄-Lax was 4.44- 7.43 nm, (c) Fe₃O₄-Lax-Eug was 6.62–13.88 nm, and that of (d) Fe₃O₄-Lax-Yla was 7.64–13.98 nm, which is comparatively lesser than that of crystallite particle size calculated with XRD technique.

Fig. 4

HRTEM graphs of A Fe₃O₄ NPs_, B Fe₃O₄-Lax, C Fe₃O₄-Lax-Eug and D Fe₃O₄-Lax-Yla

Antimicrobial activity

The functionalized magnetite materials proved a great antimicrobial effect, being active against both the Gram-positive pathogen S. Aureus and the Gram-negative pathogen E. Coli. The antimicrobial activity of the bio-active plant drugs loaded on Fe₃O₄-Lax phytohybrids was studied using common E. Coli and S. Aureus. The ZI data obtained have been summarized in Table 3.

Dynamic light scattering (DLS) studies

Particle size study was carried out using intensity distribution by DLS technique. Size distribution of Fe₃O₄ before and after surface modification was investigated in water at 25 °C. The size distribution and stability of the drug-loaded Fe₃O₄ NPs suspension was observed and recorded in Fig. 5.

Fig. 5

Size distribution analysis of drug loaded Fe₃O₄ NPs by DLS

Drug entrapment efficiency (% EE)

Drug entrapment efficiency (% EE) were analyzed using absorbance of the solution which contained the unadsorbed Eug and Yla was recorded before and after the adsorption process. The % EE values calculated, the values are given in Table 5 and the UV–Vis spectra are shown in Fig. 6.

Fig. 6

UV–Vis spectra of a Eug and b Yla pure and on Fe-Lax adsorption

In vitro free radical scavenging activity

Figure 7 illustrates spectra of antioxidant activity by Fe₃O₄-Lax-loaded Eug and Yla phytohybrids. DPPH inhibition values (%) on the Fe₃O₄ nanoconjugates are given in Table 1.

Fig. 7

Spectra of antioxidant activity by Fe₃O₄-Lax loaded Eug and Yla phytohybrids

Discussion

The focus of this investigation was to synthesize pure Fe₃O₄ NPs with Lax coating and create nanoplatforms, loaded with bioactive drug molecules of Eug and Yla. Obtaining pure conjugates in nanosize was essential, which was confirmed by studying structural morphology and particle size. Figure 1 shows X-ray powder diffraction patterns of the prepared phytohybrid nanoconjugates. The XRD pattern of Fe₃O₄ NPs could be clearly indexed with JCPDS File No. 65-3107. The lattice constant a was found to be 8.11A^o and the diffraction peaks indexed as planes with d_hkl (220), (311), (400), (422), (511) and (440) stand for cubic unit cell, characteristic and confirming the formation of cubic inverse spinel crystal system of the Fe₃O₄ structure, with the strongest reflection at (311). The absence of any characteristic peak for impurities confirmed that all the materials were pure. No shift in the peak positions in all the XRD patterns was observed, which confirms that the binding process did not result in the phase change of the magnetic Fe₃O₄ NPs [28]. The size falls in the nanometer range, confirming the formation of nanosized Fe₃O₄ NPs. Table 1 illustrates the crystallite particle size gradually decreases from 22.85 to 10.56 nm with the loading of Lax as stabilizing layer on Fe₃O₄ and further with Eug and Yla. It is also observed that in the coated Fe₃O₄ nanocomposites, the peak intensities were weakened and peak width was broadened which confirms the attachment of the coating agent on the Fe₃O₄. In drug-loaded Fe₃O₄, the peak intensity further decreases, which is attributed to the fact that the coating and drug molecules are organic in nature, giving amorphous nature to the XRD patterns, which confirms the presence of coatings. Broad diffraction peaks in the XRD pattern of coated Fe₃O₄ also indicate a further decrease in particle size after coating due to reduced agglomeration and particles tend to monodisperse.

FTIR spectra in Fig. 2 have provided structural insight into the nanoconjugates and confirm the loading of biomolecules on Fe₃O₄ NPs. The characteristic absorption peaks at 628 and 576 cm⁻¹ confirm the formation of Fe₃O₄ NPs and coated Fe₃O₄ conjugates in agreement with the literature [22]. Additional peaks and broadening are observed in drug-loaded Fe₃O₄, confirming the presence of Lax, Eug, or Yla. The weak intensity of the absorption peaks indicates the interactions between the Fe₃O₄ NPs and the coating substances are of an intermolecular type.

The H–O–H bending vibration peaks of H₂O seen in the range of 1000–1600 cm⁻¹ have a weak intensity. The Fe₃O₄ materials exhibit two characteristic peaks in the range 690 to 550 cm⁻¹, assigned to Fe–O bonding in the Fe₃O₄ crystal lattice and represent the stretching vibrations bands, related to the Fe metal in the octahedral and tetrahedral sites, respectively, in the spinel oxide structure. The peak observed at 3432 cm⁻¹ for Fe₃O₄ material relates to –OH groups and H₂O groups on the surface of Fe₃O₄ NPs, which was shifted to 3450 cm⁻¹, and 3500 cm⁻¹ in the coated samples of Fe-Lax, and Lax-Yla/Eug, respectively. The shift indicates the interaction between Lax, Eug, and Yla on the bare Fe₃O₄, in agreement with the literature [29, 30]. The peaks at 2930 cm⁻¹ and 2852 cm⁻¹ observed show the C–H stretching vibration. Some new peaks appear in the coated Fe₃O₄ and broadening of some peaks, indicate the intermolecular bonding between Lax and Fe₃O₄. When Fe₃O₄-Lax was coated with Eug & Yla, assignments are shifted for all the peaks. A shift in the Fe–O band is also observed. Thus, it could be confirmed that Lax, Eug, and Yla were attached to the Fe₃O₄ successfully. No impurity peaks were observed in any of the spectra, showing all the compounds formed were pure. The spectra were matched with standard literature data [29, 30].

Figure 3 shows the VSM spectra and the extracted parameters values of Ms, Mr and Hc are shown in Table 3. The absence of a hysteresis loop in all the graphs confirms the prepared nanoconjugates are superparamagnetic. Such materials have high Ms and zero coercivity and remanence magnetization. The magnetic moment of the particles, in the presence of magnetic field H, will align with the magnetic field direction, leading to a macroscopic magnetization of the compounds. The magnetization approached saturation when the magnetic field was increased to 15,000 Oe. The Ms values depend on the synthesis method and particle size. The Ms value of Fe₃O₄ NPs is high and matches well with standard magnetite values. The Ms values of coated Fe₃O₄ decrease, confirming the presence of coatings on the Fe₃O₄. On coating of the Fe₃O₄ NPs, the particle size decreases and thus Ms value decreases, which is attributed to the incorporation of Fe₃O₄ MNPs into Lax-Eug and Lax-Yla systems, which added a thick polymer layer on the particle surface [28]. Decreased Ms values also confirms that the particles behave as single domains. However, this amount of Ms is sufficient for biological applications of the ferrofluids in their use as drug carriers, under the application of an external magnetic field.

TEM analysis revealed that the mean core size of Fe₃O₄ NPs was 4.30–17.48 nm, Fe₃O_4-Lax was 4.44–15.43 nm, Fe₃O₄-Lax-Eug was 6.62–13.88 nm and that of Fe₃O₄-Lax-Yla was 7.64–13.98 nm, which are comparatively lesser than that of crystallite particle size calculated with XRD technique. As expected, the TEM size is smaller for the conjugate nanomaterials. Such small sizes are of great help to the superparamagnetic Fe₃O₄ drug-loaded NPs which enables the passive tissue targeting due to enhanced permeability, sustained release, and retention capacity upon administration. In addition, it is also possible to obtain the desired fine-tune ratio of hydrophilic and hydrophobic nature of conjugates, besides the whole molecular weight to manipulate the particle size and optimize the delivery efficiency [31]. The degree of agglomeration decreased after coating with Lax, Eug, and Yla, showing spherical-shaped monodispersed particles. The images show the amorphous nature of particles due to the presence of organic coatings on them.

Antimicrobial tests by the zone of inhibition (ZI) method as crucial studies were carried out to quantitatively determine the level of inhibition of the bacteria by the nanoconjugates explains the efficacy of the drug molecules under investigation. The bacteria under study were commonly found E. coli and S. aureus. It is seen in Table 3a that the ZI for Eug & Yla is 15 mm and zero, respectively, revealing that by themselves they have less or no activity against the bacteria E. Coli. The values of ZI for coated Fe₃O₄ are represented in Table 3b. It was observed that there is a synergistic increase in the antibacterial activity of the drugs Eug & Yla when loaded on Fe₃O₄-Lax. The ZI values confirm the bactericidal activity, and it is assumed that the drug-coated Fe₃O₄ are stable in the bio medium which further stabilizes the drug-loaded onto them. Therefore, the retention time between the stable coated Fe₃O₄ and the bacterium increases, which then modulates the bacterial proteins and thus arrest bacterial growth in agreement with the literature [2]. The nanoconjugates under study have shown noteworthy antimicrobial activities against gram-negative bacteria E. Coli in comparison to gram-positive S. Aureus, although the mode of action and mechanism of their antimicrobial activities have not been clarified. The stronger interactions of Eug and Yla loaded on Fe₃O₄—Lax phytohybrids on the thinner cell wall of Gram-negative bacteria of E. Coli due to the surface charge and surface energy may be responsible for the better antimicrobial activity. Thus, it was concluded that by using the combination of biocompatible coated Fe₃O₄ along with the plant-derived drugs, there was enhanced antimicrobial activity. The study says that the dosage of the drug required for any illness can be decreased by this method as the drug is entirely released at the site of infection by using an external magnetic field [10]. This proves that drug-loaded- Fe₃O₄ NPs greatly enhances the activity of the drug and can act as good drug carriers.

The amount of drug Eug and Yla loaded on Fe₃O₄ NPs was measured as drug entrapment efficiency (% EE) in the range of 200 to 800 nm. After the adsorption process, the absorption peak intensity of Eug /Yla decreases indicating that some amount of the drug is adsorbed on the Fe₃O₄-Lax conjugates. As seen in Fig. 6, the UV–Vis spectra, there was good adsorption of all the drugs on Fe₃O₄-Lax_. This indicates that the drugs were encapsulated onto Fe-Lax conjugates, with relatively high % EE values which is due to strong hydrophobic interaction between the plant drugs and the Fe-Lax conjugates. Therefore these Fe₃O₄ could act as drug carriers.

The significant increase in hydrodynamic size D(h) can be associated with the core–shell expansion after loading Fe₃O₄ with Lax, Eug, and Yla. As seen in Table 4, polydispersity index (PI) values decrease indicating high dispersion of the nanoparticles after loading with Lax extract and the drugs. The hydrodynamic size and PI values exhibit stability of Fe₃O₄ NPs in blood circulation time [16] (Table 5).

Table 4 Cummulant particle size D(h) and polydispersity index (PI)

Table 5 EE (%) value for Eug and Yla on Fe₃O₄- Lax phytohybrids

Conclusions

In conclusion, the plant-derived drug-loaded functional Fe₃O₄ NPs as a base were explored for enhanced antibacterial and antioxidant therapy, with potential advantages in the field of nanobiomedicine. Successfully synthesized Fe₃O₄ nanoconjugate materials viz. Fe₃O₄, Fe₃O₄-Lax, Fe₃O₄-Lax-Eug & Fe₃O₄-Lax-Yla were characterized for nanorange of 4.30 to 13.98 nm, spherical, monodispersed, high purity, the presence of coatings and secondary shell drug coatings and for the material's superparamagnetic nature. The antimicrobial activity show inhibition zones on the microorganisms with enhanced activity. These nanoconjugates proved to be efficient for stabilizing and controlling the release of the drugs. The results affirm that the phytohybrid derived Fe₃O₄-Lax-Eug & Fe₃O₄-Lax-Yla nanoconjugates are prototype alternatives in the exploring of new strategies to eradicate and prevent microbial infection that entail resistance.