Iron oxide nanoparticles functionalised thyme oil topical gel for anticancer and antibacterial therapies: fabrication and characterization

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Thyme oil, known for its inherent antibacterial and anticancer characteristics, is frequently constrained by its low water solubility and high volatility. The present study aimed to fabricated and characterized the thyme oil loaded iron oxide nanoparticles topical gel (TYO-FeONPs) and scrutinized its anticancer and antibacterial activity. The physicochemical features of the TYO-FeONPs, were meticulously characterised using FT-IR, FESEM and EDX electron microscopy techniques. GC-MS study exhibited the presence of ρ-Methyl salicylate (10.10), p-Cymene-2,5-diol (13.51), Caryophyllene (6.71), and Camphor (7.25). in the oil. Fe-SEM spectroscopy showed the dispersed the particle of TYO-FeONPs in clusters form with good drug encapsulation of 76.4 ± 3.127%. TYO-FeONPs gel and TYO-FeONPs demonstrated higher drug release and zone of inhibition against P.aerogeniosa and Bacillus subtilis . HaCaT cell lines were chosen to assess the cytotoxicity of TYO-FeONPs gel and 75% alive cells. This work emphasises the promise of TYO-FeONPs gel as a dual-purpose therapeutic agent for cancer treatment and bacterial infection control. iron oxide nanoparticles essential oil thyme oil anticancer antibacterial topical gel Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Since microorganisms are crucial to photosynthesis, nitrogen fixation, vitamin synthesis, and the breakdown of organic materials, human existence largely depends on their activity [ 1 ][ 2 ][ 3 ] . However, serious immunodeficiencies could result from an imbalance between the pathogenic microbes and the immune system and microbes [ 4 ] . Infectious diseases are generally caused by pathogenic bacteria, viruses, parasites, and fungi. They are directly or indirectly spread using living vectors—air, water, or food—and are responsible for millions of deaths annually worldwide. They are a major priority for health policy [ 5 ][ 6 ][ 7 ] . Microorganism-induced infections pose a threat to human health. Antibiotics are typically used to treat bacterial infections, but antibiotic resistance develops and spreads quickly for a variety of reasons, including the natural horizontal gene transference process (conjugation, transformation, and transduction), which can spread antibiotic resistance across multiple bacterial species and is primarily linked to improper antibiotic use [ 8 ] . To tackle bacterial pathogenicity, virulence factors including toxins, enzymes, and structural features like flagella, pills, capsules, etc., need to be neutralised or repressed. Virulence factors cause disease by harming or deceiving the host's immune system. This is an additional method of fighting germs and has a significantly lower chance of developing antibiotic-resistant bacteria [ 9 ] . The use of chemically produced biosynthetic nanoparticles as antimicrobial agents has grown in popularity recently. Particularly considering that the majority of plants utilised to create nanoparticles include antibacterial qualities [ 10 ] . Furthermore, microbial adherence and quick cell entrance depend on nanoparticles (NPs) with a wide surface area [ 11 ][ 12 ] . Because ferric oxide nanoparticles can interact with different bacterial compounds and stop the growth of microorganisms, they can be used to make germs less harmful and less resistant to antibiotics [ 13 ] . Essential oils (EOs) are secondary metabolites derived from plants that are becoming more and more important in defence systems [ 14 ] [ 15 ] . Since ancient times, EOs—a complex blend of volatile and odoriferous organic compounds—have been utilised in dermatology, cosmetics, and natural self-care products, among other medical fields [ 16 ] . However, it is difficult to administer EOs in a way that achieves the minimum inhibitory concentration against bacterial pathogens due to their lipophilic and volatile characteristics. Strategies involving nanotechnology and microencapsulation may provide a viable remedy in this respect [ 17 ] . Magnetic iron oxide nanoparticles are being thoroughly researched for several uses, such as drug delivery, magnetic resonance imaging, and theranostics, due to their advanced stage of use and the availability of commercial formulations formulations [ 18 ] . Their usefulness stems from the potential to use magnetic fields to guide the nanoparticles to the intended location, which will further cause heating and regulate the release of the medications. Iron oxide nanoparticles, especially magnetite, are very helpful because they are biocompatible, biodegradable, non-toxic, and can target tissue and primary structures that make it easier for different treatments to stick beneficial [ 19 ] . Because of this, systems that use magnetite nanoparticles (MNPs) and essential oils (EOs) might provide a new and useful way to treat infectious diseases disorders [ 20 ] . Thyme oil (THO) is extracted from aerial portions (fresh flowering) of Thymus vulgaris by employing steam distillation with constituents of thymol (37–55%) and carvacrol (0.5–5.5%). Thyme oil showed an excellent medicinal property. Thymol oil possesses antioxidant, spasmolytic, antifungal, antiviral, and anti-inflammatory properties, and it has shown antimicrobial activity against various strains. Particularly, when it comes to bacteria, thymol and carvacrol work similarly because they both integrate into the pathogen's lipid layer and raise its surface curvature, which alters the membrane's structure and destabilises it. They also cause changes in membrane proteins, fluidity, and elasticity elasticity [ 21 ] .Although it is lipophilic and highly volatile, exposure to high temperatures, oxygen, or light may cause deterioration [ 22 ] .. Metallic nanoparticles (NPs) can incorporate THO to overcome these issues. Therefore, this study aimed to synthesise iron oxide nanoparticles functionalised with thyme oil (TYO-FeONPs) topical gel and to evaluate their encapsulation effectiveness, particle size, and morphological features. Carbopol gel also contains these TYO-FeONPs, making it easy to administer to sick areas. So, in this study, we decided to use Carbopol gel for transdermal medication delivery. It increases the drug's biocompatibility, permeability, viscosity, flexibility, washability, skin hydration, and contact time with the sensitive skin surface [ 23 ] .The TYO-FeONPs gels that were made were also tested for their ability to kill bacteria, their rheological properties, and their ability to fight bacteria and cancer. 2. Materials and Methods 2.1 Chemical Thyme oil was purchased from Heilen Biopharm Pvt. Ltd., Carbopol 940 was procured from Ases Chemical Works, Rajasthan, triethanolamine was procured from Merck Limited, Mumbai, and methylparaben from Anant Pharmaceuticals Pvt. Ltd., Mumbai, and propylene glycol from Vizag Chemical Vishakapatnam. All the chemicals and reagents used are of standard quality in the present study. 2.2 GC–MS study of Thyme Oil Gas chromatography/mass spectrometry was employed to assess the sample of thyme oil (Shimadzu-QP-2010 ULTRA). Helium was used as a carrier gas, the injection temperature was set to 200°C, and the mode was split in the TRB-WAX column at a flow rate of 1.9 mL/min. The ionisation mode was electronic impact mode (SEI), and the oven temperature was preheated from 60°C to 260°C after three minutes with five rates and a ten-minute hold time. The chemical parts of essential oils were recorded by comparing them to mass spectra, which can be found in the NIST11s (National Institute of Standards and Technology) database library and other sources [ 24 ] . 2.3 Preparation of iron oxide nanoparticles Iron oxide nanoparticles were fabricated as per the method explained by Fatima et al. A homogeneous solution was prepared using FeSO ₄·7H₂O (0.2780 g) and ethylene glycol (5 mL). Eight millilitres of a 0.5 M KOH solution were prepared and thereafter added dropwise to a solution of 0.2780 g of FeSO₄·7H₂O in ethylene glycol while maintaining steady stirring. The resultant solution was subsequently agitated for 24 hours at 200°C to form black solids [ 25 ] . Prepared black solids were subsequently washed with an ethanol/water solution separated using magnetic separation and dried. 2.4 Physicochemical Characterisation of Nanoparticles 2.4.1 FTIR Two PerkinElmer instruments were used for FTIR spectroscopy on the topical gel mixtures to look for different functional groups in the 4000–500 cm⁻³ range. 2.4.2 Morphology of Nanoparticles and EDX The data was analysed using SEM (field emission electron microscopes; JEOL JSM-6390LV) to examine the morphology and architecture of the TYO-FeONPs. Double-sided adhesives were used to mount the samples on copper tubes in preparation for this. An FESEM with gold was then used to analyse each sample for 120 seconds at a current of 20 mA. 50 kV was the voltage that was employed. was employed [ 26 ][ 27 ] . EDX was employed to detect the presence of elemental in the nanoparticles. 2.4.3 Determination of % Drug Entrapment Efficiency The ultrafiltration method was used to assess the DEE% of the produced TYO-FeONPs by the procedure outlined in the literature [ 28 ] . In ultracentrifuge filter tubes, the formulations (2 mL) were centrifuged for 20 to 30 minutes at 25°C and 3000 rpm, and the unconfined drugs were removed from the tubes with the help of centrifugal forces. The UV technique was used to measure the unconfined TYO using the following formula: % 𝐃rug entrapment efficiency= (𝐓𝐨𝐭𝐚𝐥 𝐚𝐦𝐨𝐮𝐧𝐭 𝐨𝐟 TYO−𝐀𝐦𝐨𝐮𝐧𝐭 𝐨𝐟 TYO 𝐢𝐧 𝐬𝐮pernatant)/ Total amount of TYO X 100 2.5 Preparation of thyme oil-loaded nanoparticle gel ( TYO-FeONPs) topical gel A 1:1 mix of sodium alginate and carbopol was mixed in 10 ml of distilled water and stirred at 200–600 rpm all the time until a clear gel was formed. THO was transferred to the above gel with continuous agitation at room temperature along with Carbopol (gelling agent) and surfactant/emulsifier to help in the dispersion and formation of a gel emulsion with the addition of Methylparaben (preservative). Fabricated iron oxide nanoparticles were added to the produced gel emulsion while maintaining stirring for 20 minutes until converted to gel. In the formulated topical gel, propylene glycol was used as a permeation enhancer, and triethylamine was added to adjust the pH. The produced topical gel formulations, TYO-FeONPs topical gel, were kept aside for subsequent examination and fabricated as per Table 1 . Table 1 Formulation of thyme oil loaded iron oxide nanoparticles topical gel Sr. No. Ingredients Sodium Alginate Carbopol 940 Thyme Oil Iron oxide Fe 3 O 4 Nanoparticles Methylparaben 1. F 1 100mg 100mg 2ml 0.18g 2. F 2 100mg 100mg 2ml 50mg 0.18g 3. F 3 100mg 100mg 2ml 60mg 0.18g 4. F 4 100mg 100mg 2ml 70mg 0.18g 5. F 5 100mg 100mg 2ml 80mg 0.18g 2.6 Evaluation of TYO-FeONP's topical gel 2.6.1 Organoleptic performance : The colour of the topical gel formulation was examined using a white background. The odour was checked by placing the topical gel in water and checking the smell. The gel's greasiness was checked by applying the formulation to the skin. 2.6.2 Rheological Study Viscosity TYO-FeONPs gels were pre-experimented and stored at room temperature. We tested viscosity using the Brookfield viscosity meter. Processing the gel formulation at moderate rates does not harm the gel structure [ 29 ] .. pH The pH of 0.5 g of gel was measured after it was weighed and dissolved in 50 mL of pure water using a digital pH meter [ 29 ] . Spreadability The 0.5 g gel was pressed using horizontal plates of 20 x 20 cm. Five minutes or so were then spent with a 500 g weight on the top plate. Centimetres are used to express the dispersion circle's diameter [ 30 ] . Average values were calculated as a triplicate of results, which were performed separately. We calculated it using the following formula S = M.L/T S represents the spreadability in grammes.cm/sec, M is the mass in grams, T is the time 2.6.3 In vitro Drug Release Studies An in vitro drug release investigation of TYO-FeONPs gels (F1 to F5) were performed in the dialysis bag approach at 37°C in PBS with a pH of 6.8. The dialysis bag contained all formulation gels and were dialysed in a shaking incubator containing 40 ml of phosphate buffer solution at room temperature. After that, an aliquot (3 ml) was taken at predetermined intervals. As soon as the PBS was taken out, enough fresh PBS was injected to maintain the sink's functionality [ 31 ] . The amount of TYO released from the TYO-FeONPs gel was measured at a wavelength of 260 nm and conducted in triplicate. 2.6.4 Antimicrobial activity assay Bacterial culture (P. aeruginosa, MTCC-3541) and B. subtilis (MTCC 1133) were purchased from the Microbial Type Culture Collection and Gene Bank (MTCC) in Chandigarh. The zone inhibition method was used to assay the antibacterial activity following the Kirby-Bauer method [ 32 ] . The MHA plates were inoculated by spreading 100 µl of P. aeruginosa, B. subtilis, and bacterial culture (the inoculum was made by adjusting 0.5 McFarland units—approximately 1.5 x 108 CFU/mL from Mueller-Hilton broth). The discs with 10 µl of various concentrations (0 to 100 mg/ml) were then placed on top of the inoculum. The positive control was the ciprofloxacin disc (10 µg) in each plate, while the vehicle control was one disc supplied with solvent only. The plates of P. aeruginosa and B. subtilis were incubated (Basil Scientific Corp. India—Incubator) for 24 hrs at 37°C. A clear zone created around the disc was checked and noted. 2.6.5 Time-Kill Kinetics Assay The TYO-FeONPs gel time-kill kinetics were performed using the method outlined by [ 33 ] .. Concentrations that were three times the TYO-FeONPs gel's MIC were created. Sterile water was transferred to bacterial broth, whereas the negative control was test samples. For twenty-four hours, the bacterial cells' vitality was continually observed. Samples were serially diluted in PBS after being collected at predetermined intervals. At 37°C, they were incubated for the entire night. Bacterial growth in the colonies on the plates was measured after the incubation period. We plotted the graph against the time after calculating and converting the colony-forming unit (CFU) to log CFU/mL. 2.6.6 MTT Assay As per the procedure described by [39, 42], the cytotoxicity of samples on the HaCaT cell line (procured from NCCS Pune) was carried out by MTT assay. 10% foetal bovine serum (FBS) was added to the DMEM (Dulbecco's modified Eagle's medium), which was the medium used to cultivate the cells. The cells were kept in an incubator with 5% carbon dioxide and humidity at 37°C. Cell survival was measured using an MTT assay. The experiment involved 100 µL of suspension, which contained 1 × 104 cancer cells in 96-well plates, and cells were treated with different concentrations of TYO-FeONPs for a further 24 hours. 5 mg/mL of working MTT solution (20 µL) was added to each well and incubated for 4 hours. Untreated cells were termed as control, whereas cells not treated with MTT were blank. We used an Elisa plate reader (iMark, Biorad, USA) to note the absorbance at 540nm [ 34 ] . % 𝐂𝐞𝐥𝐥 𝐯𝐢𝐚𝐛𝐢𝐥𝐢𝐭𝐲= 𝐀540 𝐧𝐦 𝐭𝐫𝐞𝐚𝐭𝐞𝐝 𝐜ells/A540 nm untreated cells X 100 2.6.7 Stability Study Through short-term changes in physical stability, we measured the impact of temperature on the TYO-FeONPs gel and kept it for 0, 15, and 30 days at ambient temperature as per the procedure suggested by ICH guidelines. We examined the samples for physicochemical characteristics, such as pH, %DEE, colour, and particle size, at each interval [ 35 ] . 3. Result 3.1 Essential Oil Composition GC/MS was used to determine the chemical composition of the essential oil. The results of this analysis are presented in Fig. 1 and Fig. 2 and resulted in the identification of eighty-four bioactive constituents, which shows 100% of total oil. 2,4-Bis(diazo)adamantane, which accounted for 43.67% of the chemicals discovered, was determined to be the most prominent agent, followed by ρ-Methyl salicylate (10.10), p-Cymene-2,5-diol (13.51), Caryophyllene (6.71), and Camphor (7.25). In addition to this, other terpenes are also present in small amounts, such as Borneol, Camphene, p-Cymen-7-ol, etc [ 36 ] .. 3.2 Characterisation of thyme-loaded iron oxide nanoparticles 3.2.1 FTIR: Figure 3a and b shows the FTIR spectra of the thyme oil and thyme-loaded iron oxide nanoparticles (TYO-FeONPs). The O–H stretch caused the absorption peak in the pure thyme oil at 3239 cm⁻¹. The aliphatic C–H stretch explains the sharp peak at 2332 cm.=O carbonyl group is responsible for the peak at 1635 cm⁻¹; further evidence of an ester component comes from the C–O stretch bend seen at 1247 cm⁻¹ [ 37 ] . The C–H deformations are reflected in the peaks at 787, 1103, and 1247 cm⁻¹. The formulation's absorption peaks, displayed in fig. b, did not significantly differ. 3.2.2 Particle size and shape of TYO-FeONPs The FE-SEM image of TYO-FeONPs is displayed in Fig. 4a and b. The particles in the TYO-FeONPs are dispersed in clusters of various forms, as demonstrated by the microscopic FE-SEM image. Given that it was in great agreement with previously published results, which explain that the population was homogeneous, it is possible to conclude that the optimal particle size of the formulation TYO-FeONPs was satisfactory. The elemental composition of metal nanoparticles is detected using energy dispersive spectroscopy (EDS), which also separates the unique X-rays of various elements into an energy spectrum. The EDS spectra displayed in Fig. 4c corroborate the existence of the Fe and O elements. The use of thyme oil in the production process serves as a capping agent around the nanoparticles and provides stability, so the carbon element exists. 3.2.3 Percentage Drug Entrapment Efficiency of Thyme-loaded Nanoparticles As per encapsulation efficiency measurement, TYO-FeONPs had an encapsulation effectiveness of 76.4 ± 3.127% and oil was highly encapsulated. The saturation of the polymer dispersion caused the encapsulation efficiency to reach its maximum and thus remain constant. 3.3 Characterisation of TYO-FeONPs Gel 3.3.1. Physical Parameters: The physical parameters of the prepared formulation and nanoparticles is shown in table no 1. Iron oxide nanoparticle gel were found to be dark black colour with fine consistent and odourless whereas thyme oil loaded iron oxide nanoparticle gel was yellowish brown in colour with greasiness and aromatic odour. Table 1 Physical Parameters Color Consistency Greasiness Odour Fe 3 O 4 Dark brown /Black Fine Smooth powder Odorless Thyme oil loaded with Fe 3 O 4 Gel Yellowish Brown Consistent Slippery Aromatic 3.3.2 Rheological profile of the TYO-FeONPs topical gel The gel was scrutunized for the pH, viscosity and spreadability and presented in table. 2. We fabricated a TYO-FeONPs topical gel that may be applied on the skin's surface to achieve consistent dose forms and superior active ingredient carriers. With a pH of 6.2 ± 0.1 to 6.5 ± 0.4, the TYO-FeONPs gel is ideal for topical administration and does not irritate the skin and formulation F3 had a pH of 6.5 ± 0.4. The viscosity was measured at 34,123 cps to cps to 38,142 cps for tested the fabricated gel's spreadability. Between 35,000 and 40,000 cps is the ideal viscosity for topical preparation because it offers spreadability and acceptable flow characteristics and all fabricated formulation shown lower viscosity in comparision to pure thyme oil gel and unveiled shear thining behaviour [ 38 ] . 9.426 ± 2.002 cm was the measured spread distance of the formulation F3. An acceptable spreadability value was attained after determining the gel's viscosity. According to reports in the literature, the topical gel's exceptional viscosity and spreadability make it simple to apply a layer to the skin [ 39 ] . Table 2 Rheological profile of the TYO-FeONPs topical gel Sr. No. Ingredients Thyme Oil Iron oxide Fe 3 O 4 Nanoparticles Spreadability pH viscosity 1. F 1 blank gel 2ml - 7.76 ± 1.982 cm 6.2 ± 0.1 36,146 cps 2. F 2 2ml 50mg 8.43 ± 1.876 cm 6.4 ± 0.3 34,123 cps 3. F 3 2ml 60mg 9.426 ± 2.002 cm 6.5 ± 0.4 32,201 cps 4. F 4 2ml 70mg 8.94 ± 2.323 cm 6.3 ± 0.2 34,142 cps 5. F 5 2ml 80mg 9.02 ± 1.093 cm 6.2 ± 0.1 35,221 cps 3.3.3 In Vitro Drug Release Profile of TYO-FeONPs Topical Gel In vitro drug release profile of pure thyme oil (TYO), thyme oil loaded iron oxide nanoparticles (TYO-FeONPs) and thyme oil loaded iron oxide nanoparticles gel (TYO-FeONPs gel) were presented in Fig. 5 . And buffer solution (pH 6.8) were selected as a medium and amount of drug release were evaluated. As per result, we noted that pure thyme oil gel release 56% of drug within 4 hours as comparison to TYO-FeONPs and TYO-FeONPs gel. We found that 87.05% of drug were released within 8 hrs (thyme oil gel), whereas TYO-FeONPs were released only 78.58% and TYO-FeONPs gel released 75.48% in a buffer solution. The drug release was significantly more in case of TYO-FeONPs gels as compare to other formulation. 3.3.4 In Vitro Antimicrobial Activity Figure 6a and b and Table 3 displayed the antibacterial activity of the TYO-FeONPs, TYO-FeONPs gel and standard drug ciprofloxacin against B.subtilis and P.Aeruginosa . The topical gel formulations containing thyme oil-loaded iron oxide nanoparticles at a concentration of 60 mg Fe₃O₄ exhibited favourable physicochemical features and superior content relative to alternative formulations. Consequently, these formulations were subsequently chosen for antibacterial investigations. The findings of antimicrobial investigations indicated that the topical gel formulation of thyme oil effectively inhibits microbes during wound healing as reported in the literature [ 40 ][ 41 ] . It was observed that the TYO-FeONPs shown higher antibacterial activity than the TYO-FeONPs gel against gram positive ( B. subtilis ) and gram negative bacteria P. aeruginosa the most common resistant strains which is responsible for sepsis, infection and chronic wound [ 42 ][ 43 ] . The capability of gels to shown higher antibacterial activity due to various reasons such as interaction between the drug carrier, nano size of formulation, occlusive property [ 44 ] . The wide range of microbial activity against B. subtilis and P. aeruginosa caused by the encapsulation of iron oxide nanoparticles in the gel (TYO-FeONPs gel) may be the cause. The formulations are promising medicines for treating infected wounds, as evidenced by their antibacterial efficacy. Table 3 Zones of inhibition (mm) of TYO-FeONPs and TYO-FeONPs gel against test organisms Organism Positive control (mm) TYO-FeONPs TYO-FeONPs gel B. subtilis 24.45 8.5 8.9 P. Aeruginosa 20.34 8.4 8.7 Proposed mechanism for antibacterial activity of TYO-FeONPs and TYO-FeONPs gel Through direct contact with the environment, TYO-FeONPs and TYO-FeONPs gel demonstrated antibacterial efficacy against the test microorganism strains and samples were found to be effective promising antibacterial agent The inhibition zone significantly found to be higher when treated with TYO-FeONPs gel topical gel. The proposed mechanism that may be connected to the thyme oil-loaded iron oxide nanoparticles is based on, i.e., iron oxide nanoparticles combat bacteria through oxidative stress by the production of reactive oxygen species and by damaging cell walls and membrane via electrostatic and van der Waals interactions. Bacterial membranes bind metal cations via highly electronegative chemical groups [ 45 ] . Due to their positive charge, metal oxides stick to negatively charged bacteria [ 46 ] . Electrostatic and van der Waals interactions allow iron oxide nanoparticles to stick to bacterial cell walls and membranes [ 47 ] . Next, iron oxide nanoparticles cluster on the bacterial surface and destroy cell walls and membranes, leaking bacterial material [ 48 ] . During fenton reaction there is the ROS production like nonradical hydrogen peroxide, hydroxyl radical and superoxide anion and induced oxidative stress [ 49 ] . The antibacterial activity by iron nanoparticles inside the cell wall by the release of iron ions and damage the DNA, lipids, and proteins directly, leading to oxidative damage and bacterial death [ 13 ] . The antibacterial activity is depending on bacteria which converts ferric ions to ferrous and, suggesting that ROS generation was their principal antibacterial mechanism [ 50 ] . The research found that essential oils and iron oxide nanoparticles work together to kill tested bacterial strains and shown in Fig. 7 . 3.3.5. Time-Kill Assays Figure 8 displays the rates at which TYO-FeONPs gel and TYO-FeONPs killed gram-positive bacteria Bacillus subtilis over the course of 24 hours at 37°C. After a few hours, the result showed that TYO-FeONPs and TYO-FeONPs gel formulation and gel showed a log decrease, indicating a quick bactericidal impact. 3.3.6. In Vitro Cell Viability Assay As illustrated in Fig. 9 , HaCaT cell lines were chosen to assess the cytotoxicity of TYO-FeONPs gel. The results showed that after being grown in TYO-FeONPs gels, more than 75% of the cells were still alive. These outcomes demonstrate that the iron oxide loaded thyme oil gel has no negative consequences and that the cells can grow properly. The fact that TYO-FeONPs gel is not harmful to living things and works well with them in these studies shows how useful they could be for in vitro uses. 2.2.6: Stability Study Drugs' long-term stability is crucial, yet it can be challenging to attain, particularly in liquid forms. The stability investigation of the TYO-FeONPs gel is displayed in Table 4 . TYOs instability due to heat, air, and light was expected to be reduced by encapsulating it in TYO-FeONPs gel. Our study's findings indicate that breakdown and volatility cause a considerable loss of oil components. Table 4 Stability study of TYO-FeONPs gel at ambient temperature Days Colour Texture consistency pH 0 White Smooth Consistent 6.0 15 White Smooth Consistent 6.0 30 white smooth Consistent 6.0 Conclusion The study successfully synthesised and characterised iron oxide nanoparticles functionalised with thyme oil for dual anticancer and antibacterial purposes. Oil with iron oxide nanoparticles significantly improved its solubility and stability, hence increasing its medicinal potential. The TYO-FeONPs gel showed strong antibacterial efficacy against multiple bacterial strains and showed considerable cytotoxicity towards cancer cells, triggering apoptosis and could be used as a versatile medicine. To make it easier for these nanoparticles to be used in the future, more research needs to be done on them in living things and on how safe and effective they are over time. Declarations Acknowledgement: We are also thankful to the SHUATS, Prayagraj for the continuous support and motivation and thankful to Aakar biotechnology, Lucknow for in vitro anticancer and in vitro antimicrobial study and Panjab University, Bathinda for instrumentation. The author are extremely thankful to biorender (https://www.biorender.com/) for the illustration. Author Contributions AS conceptualized, formal analysis and methodology the manuscript; DS review and editing the manscript; AV and DS writing original draft preparation. All authors have read and agreed to the published version of the manuscript. Funding None Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. 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V. De Oliveira Brisola MacIel, W. G. Sganzerla, A. Da Rosa Almeida, R. D. De Armas, M. H. MacHado, C. G. Da Rosa, M. R. Nunes, F. C. Bertoldi, P. L. M. Barreto, Mater. Res. Express 2020 , DOI 10.1088/2053-1591/ab6c63. Y. H. Lin, J. H. Lin, Y. S. Hong, J. Biomed. Mater. Res. - Part B Appl. Biomater. 2017 , DOI 10.1002/jbm.b.33394. Y. Li, J. Wang, Y. Yang, J. Shi, H. Zhang, X. Yao, W. Chen, X. Zhang, Mater. Sci. Eng. C 2021 , DOI 10.1016/j.msec.2020.111447. R. Augustine, S. R. U. Rehman, R. Ahmed, A. A. Zahid, M. Sharifi, M. Falahati, A. Hasan, Int. J. Biol. Macromol. 2020 , DOI 10.1016/j.ijbiomac.2020.03.207. Y. M. Zhang, C. O. Rock, Nat. Rev. Microbiol. 2008 , DOI 10.1038/nrmicro1839. B. Li, B. E. Logan, Colloids Surfaces B Biointerfaces 2004 , DOI 10.1016/j.colsurfb.2004.05.006. L. Shkodenko, I. Kassirov, E. Koshel, Microorganisms 2020 , DOI 10.3390/microorganisms8101545. Y. Li, D. Yang, S. Wang, C. Li, B. Xue, L. Yang, Z. Shen, M. Jin, J. Wang, Z. Qiu, Molecules 2018 , DOI 10.3390/molecules23030606. Q. Tian, F. Xue, Y. Wang, Y. Cheng, L. An, S. Yang, X. Chen, G. Huang, Nano Today 2021 , DOI 10.1016/j.nantod.2021.101162. S. V. Gudkov, D. E. Burmistrov, D. A. Serov, M. B. Rebezov, A. A. Semenova, A. B. Lisitsyn, Antibiotics 2021 , DOI 10.3390/antibiotics10070884. Additional Declarations No competing interests reported. Supplementary Files floatimage1.jpeg Graphical abstract Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7207816","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":507255858,"identity":"ee7ae1b9-5cd8-4416-9a80-3db03d49b4be","order_by":0,"name":"Abhayjeet Singh","email":"","orcid":"","institution":"Sam Higginbottom University of Agriculture, Technology and Sciences","correspondingAuthor":false,"prefix":"","firstName":"Abhayjeet","middleName":"","lastName":"Singh","suffix":""},{"id":507255859,"identity":"3bb69ac6-0156-4688-958d-c087c3ac004d","order_by":1,"name":"Amita Verma","email":"","orcid":"","institution":"Sam Higginbottom University of Agriculture, Technology and Sciences","correspondingAuthor":false,"prefix":"","firstName":"Amita","middleName":"","lastName":"Verma","suffix":""},{"id":507255860,"identity":"4e85b4e1-48a8-4227-9ee2-76deab9c8b1d","order_by":2,"name":"Deepika Singh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDCCA0CcAEQGIPYHIMHGTrwWZoaDM0BamInRwgDVwswDYhLSwne8+diDBwx2eebs/QcP2/zaJs/HzMD44WMObi2SZ46lGyQwJBdb9hxmOJzbd9uwDWib5MxtuLUY3Mgxk0hgOJC44UYyUEvPbUagFjZmXnxa7r//htBi2XPbnrCWGzxsCC0MP24nEtQieSbN3CDBIDlxw5nDBgd7G24ntzEzNuP1C9/xw88e/qiwS9xwvPHxhx9/btvOb28++OEjHi1AwAZ0HpTJ2AYmG/Cqh2iBgz+EFI+CUTAKRsFIBABif1fbDGd44gAAAABJRU5ErkJggg==","orcid":"","institution":"Sam Higginbottom University of Agriculture, Technology and Sciences","correspondingAuthor":true,"prefix":"","firstName":"Deepika","middleName":"","lastName":"Singh","suffix":""}],"badges":[],"createdAt":"2025-07-24 17:23:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7207816/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7207816/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90502510,"identity":"01566c95-e990-405f-9b54-966746f305c4","added_by":"auto","created_at":"2025-09-03 11:59:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":84146,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGC –MS of thyme oil\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7207816/v1/98c011fd5dc60254c89c5bf2.png"},{"id":90502511,"identity":"770160ef-0544-4f41-adef-35e421e4db55","added_by":"auto","created_at":"2025-09-03 11:59:02","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":534687,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage composition and chemical compounds analysis of thyme oil by GC-MS\u003c/p\u003e","description":"","filename":"2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7207816/v1/428fcad2be866f8c3934ef6b.jpeg"},{"id":90502512,"identity":"6ef61af7-8364-4040-bf73-35198f20803e","added_by":"auto","created_at":"2025-09-03 11:59:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea Graph of FTIR of Thyme oil-loaded iron oxide nanoparticle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eb \u003cstrong\u003eGraph of FTIR of Thyme oil\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7207816/v1/5a968b579aa45daafa022fe7.jpg"},{"id":90503037,"identity":"4c674707-50d4-45e5-97b2-dacdaaadcf94","added_by":"auto","created_at":"2025-09-03 12:07:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":366527,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea and b FESEM image \u003c/strong\u003eof \u003cstrong\u003eTYO-FeONPs \u003c/strong\u003ec. \u003cstrong\u003eEDX of\u003c/strong\u003e \u003cstrong\u003eTYO-FeONPs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7207816/v1/7c31d09bf1459b3482e60c93.png"},{"id":90502514,"identity":"b67e64f1-0861-4cea-bece-7a5bc6f6d389","added_by":"auto","created_at":"2025-09-03 11:59:02","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":71033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ein vitro drug release study of TYO, TYO-FeONPs and TYO-FeONPs gel in a buffer solution of pH 6.8\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7207816/v1/e959f5c4c0db9904a98edc9b.jpeg"},{"id":90502518,"identity":"e9ca54dd-7755-4c11-8f26-c5a685b5fc96","added_by":"auto","created_at":"2025-09-03 11:59:03","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":608212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea. Antibacterial activity of TYO-FeONPs and TYO-FeONPs gel on test organism\u003c/strong\u003e \u003cstrong\u003eTest organism- \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. subtilis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by Disc Diffusion and b. Test organism- \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. Aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by Disc Diffusion\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7207816/v1/5ae0e5e1c75825a8d3962ba6.jpeg"},{"id":90503040,"identity":"353676bc-f8f6-4bab-b5f3-8610dc19a95d","added_by":"auto","created_at":"2025-09-03 12:07:03","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1129108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eProposed mechanism for antibacterial activity of TYO-FeONPs and TYO-FeONPs gel\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7207816/v1/f2c774078862fb6b84b6a55a.jpeg"},{"id":90502523,"identity":"7673d4fb-59fb-40a9-a072-25bc355f095e","added_by":"auto","created_at":"2025-09-03 11:59:03","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":61874,"visible":true,"origin":"","legend":"\u003cp\u003eTime-kill study of the formulation\u003c/p\u003e","description":"","filename":"8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7207816/v1/d5692740e9adae1cfb8ed93f.jpeg"},{"id":90503041,"identity":"76e56ffc-d9fd-41f3-a0b3-5af2c221029f","added_by":"auto","created_at":"2025-09-03 12:07:03","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":16009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ein vitro cell viability assay of FEO–PLGANPs gel\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7207816/v1/c819d02b220eab1304df20a1.png"},{"id":92388039,"identity":"76c5c4f4-af44-4674-ba3d-c742480ee6bc","added_by":"auto","created_at":"2025-09-29 08:02:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4674454,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7207816/v1/c2af0a14-9a53-4e41-ae1e-679e0010f67b.pdf"},{"id":90502516,"identity":"9a352f6b-8500-47df-a976-24f04801ddd3","added_by":"auto","created_at":"2025-09-03 11:59:03","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1173627,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7207816/v1/a4a0e4e11e722513ea224d63.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Iron oxide nanoparticles functionalised thyme oil topical gel for anticancer and antibacterial therapies: fabrication and characterization","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSince microorganisms are crucial to photosynthesis, nitrogen fixation, vitamin synthesis, and the breakdown of organic materials, human existence largely depends on their activity\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e][\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. However, serious immunodeficiencies could result from an imbalance between the pathogenic microbes and the immune system and microbes \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Infectious diseases are generally caused by pathogenic bacteria, viruses, parasites, and fungi. They are directly or indirectly spread using living vectors\u0026mdash;air, water, or food\u0026mdash;and are responsible for millions of deaths annually worldwide. They are a major priority for health policy \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e][\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMicroorganism-induced infections pose a threat to human health. Antibiotics are typically used to treat bacterial infections, but antibiotic resistance develops and spreads quickly for a variety of reasons, including the natural horizontal gene transference process (conjugation, transformation, and transduction), which can spread antibiotic resistance across multiple bacterial species and is primarily linked to improper antibiotic use \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. To tackle bacterial pathogenicity, virulence factors including toxins, enzymes, and structural features like flagella, pills, capsules, etc., need to be neutralised or repressed. Virulence factors cause disease by harming or deceiving the host's immune system. This is an additional method of fighting germs and has a significantly lower chance of developing antibiotic-resistant bacteria \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe use of chemically produced biosynthetic nanoparticles as antimicrobial agents has grown in popularity recently. Particularly considering that the majority of plants utilised to create nanoparticles include antibacterial qualities \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Furthermore, microbial adherence and quick cell entrance depend on nanoparticles (NPs) with a wide surface area \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e][\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Because ferric oxide nanoparticles can interact with different bacterial compounds and stop the growth of microorganisms, they can be used to make germs less harmful and less resistant to antibiotics \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEssential oils (EOs) are secondary metabolites derived from plants that are becoming more and more important in defence systems \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Since ancient times, EOs\u0026mdash;a complex blend of volatile and odoriferous organic compounds\u0026mdash;have been utilised in dermatology, cosmetics, and natural self-care products, among other medical fields \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. However, it is difficult to administer EOs in a way that achieves the minimum inhibitory concentration against bacterial pathogens due to their lipophilic and volatile characteristics. Strategies involving nanotechnology and microencapsulation may provide a viable remedy in this respect \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Magnetic iron oxide nanoparticles are being thoroughly researched for several uses, such as drug delivery, magnetic resonance imaging, and theranostics, due to their advanced stage of use and the availability of commercial formulations formulations \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Their usefulness stems from the potential to use magnetic fields to guide the nanoparticles to the intended location, which will further cause heating and regulate the release of the medications. Iron oxide nanoparticles, especially magnetite, are very helpful because they are biocompatible, biodegradable, non-toxic, and can target tissue and primary structures that make it easier for different treatments to stick beneficial \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Because of this, systems that use magnetite nanoparticles (MNPs) and essential oils (EOs) might provide a new and useful way to treat infectious diseases disorders \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThyme oil (THO) is extracted from aerial portions (fresh flowering) of Thymus vulgaris by employing steam distillation with constituents of thymol (37\u0026ndash;55%) and carvacrol (0.5\u0026ndash;5.5%). Thyme oil showed an excellent medicinal property. Thymol oil possesses antioxidant, spasmolytic, antifungal, antiviral, and anti-inflammatory properties, and it has shown antimicrobial activity against various strains. Particularly, when it comes to bacteria, thymol and carvacrol work similarly because they both integrate into the pathogen's lipid layer and raise its surface curvature, which alters the membrane's structure and destabilises it. They also cause changes in membrane proteins, fluidity, and elasticity elasticity \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.Although it is lipophilic and highly volatile, exposure to high temperatures, oxygen, or light may cause deterioration \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.. Metallic nanoparticles (NPs) can incorporate THO to overcome these issues.\u003c/p\u003e\u003cp\u003eTherefore, this study aimed to synthesise \u003cem\u003eiron oxide nanoparticles functionalised with thyme oil\u003c/em\u003e (TYO-FeONPs) topical gel and to evaluate their encapsulation effectiveness, particle size, and morphological features. Carbopol gel also contains these TYO-FeONPs, making it easy to administer to sick areas. So, in this study, we decided to use Carbopol gel for transdermal medication delivery. It increases the drug's biocompatibility, permeability, viscosity, flexibility, washability, skin hydration, and contact time with the sensitive skin surface \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.The TYO-FeONPs gels that were made were also tested for their ability to kill bacteria, their rheological properties, and their ability to fight bacteria and cancer.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Chemical\u003c/h2\u003e\u003cp\u003eThyme oil was purchased from Heilen Biopharm Pvt. Ltd., Carbopol 940 was procured from Ases Chemical Works, Rajasthan, triethanolamine was procured from Merck Limited, Mumbai, and methylparaben from Anant Pharmaceuticals Pvt. Ltd., Mumbai, and propylene glycol from Vizag Chemical Vishakapatnam. All the chemicals and reagents used are of standard quality in the present study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 GC\u0026ndash;MS study of Thyme Oil\u003c/h2\u003e\u003cp\u003eGas chromatography/mass spectrometry was employed to assess the sample of thyme oil (Shimadzu-QP-2010 ULTRA). Helium was used as a carrier gas, the injection temperature was set to 200\u0026deg;C, and the mode was split in the TRB-WAX column at a flow rate of 1.9 mL/min. The ionisation mode was electronic impact mode (SEI), and the oven temperature was preheated from 60\u0026deg;C to 260\u0026deg;C after three minutes with five rates and a ten-minute hold time. The chemical parts of essential oils were recorded by comparing them to mass spectra, which can be found in the NIST11s (National Institute of Standards and Technology) database library and other sources \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Preparation of iron oxide nanoparticles\u003c/h2\u003e\u003cp\u003eIron oxide nanoparticles were fabricated as per the method explained by Fatima et al. A homogeneous solution was prepared using FeSO\u003csub\u003e₄\u0026middot;7H₂O\u003c/sub\u003e (0.2780 g) and ethylene glycol (5 mL). Eight millilitres of a 0.5 M KOH solution were prepared and thereafter added dropwise to a solution of 0.2780 g of FeSO₄\u0026middot;7H₂O in ethylene glycol while maintaining steady stirring. The resultant solution was subsequently agitated for 24 hours at 200\u0026deg;C to form black solids \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Prepared black solids were subsequently washed with an ethanol/water solution separated using magnetic separation and dried.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Physicochemical Characterisation of Nanoparticles\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1 FTIR\u003c/h2\u003e\u003cp\u003eTwo PerkinElmer instruments were used for FTIR spectroscopy on the topical gel mixtures to look for different functional groups in the 4000\u0026ndash;500 cm⁻\u0026sup3; range.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2 Morphology of Nanoparticles and EDX\u003c/h2\u003e\u003cp\u003eThe data was analysed using SEM (field emission electron microscopes; JEOL JSM-6390LV) to examine the morphology and architecture of the TYO-FeONPs. Double-sided adhesives were used to mount the samples on copper tubes in preparation for this. An FESEM with gold was then used to analyse each sample for 120 seconds at a current of 20 mA. 50 kV was the voltage that was employed. was employed \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e][\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. EDX was employed to detect the presence of elemental in the nanoparticles.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3 Determination of % Drug Entrapment Efficiency\u003c/h2\u003e\u003cp\u003eThe ultrafiltration method was used to assess the DEE% of the produced TYO-FeONPs by the procedure outlined in the literature \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In ultracentrifuge filter tubes, the formulations (2 mL) were centrifuged for 20 to 30 minutes at 25\u0026deg;C and 3000 rpm, and the unconfined drugs were removed from the tubes with the help of centrifugal forces. The UV technique was used to measure the unconfined TYO using the following formula:\u003c/p\u003e\u003cp\u003e\u003cb\u003e% \u0026#119811;rug entrapment efficiency= (\u0026#119827;\u0026#119848;\u0026#119853;\u0026#119834;\u0026#119845; \u0026#119834;\u0026#119846;\u0026#119848;\u0026#119854;\u0026#119847;\u0026#119853; \u0026#119848;\u0026#119839; TYO\u0026minus;\u0026#119808;\u0026#119846;\u0026#119848;\u0026#119854;\u0026#119847;\u0026#119853; \u0026#119848;\u0026#119839; TYO \u0026#119842;\u0026#119847; \u0026#119852;\u0026#119854;pernatant)/ Total amount of TYO X 100\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.5 Preparation of thyme oil-loaded nanoparticle gel\u003c/b\u003e (\u003cb\u003eTYO-FeONPs) topical gel\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eA 1:1 mix of sodium alginate and carbopol was mixed in 10 ml of distilled water and stirred at 200\u0026ndash;600 rpm all the time until a clear gel was formed. THO was transferred to the above gel with continuous agitation at room temperature along with Carbopol (gelling agent) and surfactant/emulsifier to help in the dispersion and formation of a gel emulsion with the addition of Methylparaben (preservative). Fabricated iron oxide nanoparticles were added to the produced gel emulsion while maintaining stirring for 20 minutes until converted to gel. In the formulated topical gel, propylene glycol was used as a permeation enhancer, and triethylamine was added to adjust the pH. The produced topical gel formulations, TYO-FeONPs topical gel, were kept aside for subsequent examination and fabricated as per Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFormulation of thyme oil loaded iron oxide nanoparticles topical gel\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSr. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIngredients\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSodium\u003c/p\u003e\u003cp\u003eAlginate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCarbopol\u003c/p\u003e\u003cp\u003e940\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eThyme\u003c/p\u003e\u003cp\u003eOil\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIron oxide\u003c/p\u003e\u003cp\u003eFe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eNanoparticles\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMethylparaben\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.18g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e50mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.18g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e60mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.18g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e70mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.18g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e80mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.18g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Evaluation of TYO-FeONP's topical gel\u003c/h2\u003e\u003cp\u003e\u003cb\u003e2.6.1 Organoleptic performance\u003c/b\u003e: The colour of the topical gel formulation was examined using a white background. The odour was checked by placing the topical gel in water and checking the smell. The gel's greasiness was checked by applying the formulation to the skin.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2 Rheological Study\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eViscosity\u003c/strong\u003e\u003cp\u003eTYO-FeONPs gels were pre-experimented and stored at room temperature. We tested viscosity using the Brookfield viscosity meter. Processing the gel formulation at moderate rates does not harm the gel structure\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e..\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003cp\u003eThe pH of 0.5 g of gel was measured after it was weighed and dissolved in 50 mL of pure water using a digital pH meter \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSpreadability\u003c/strong\u003e\u003cp\u003eThe 0.5 g gel was pressed using horizontal plates of 20 x 20 cm. Five minutes or so were then spent with a 500 g weight on the top plate. Centimetres are used to express the dispersion circle's diameter \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Average values were calculated as a triplicate of results, which were performed separately. We calculated it using the following formula\u003c/p\u003e\u003c/p\u003e\u003cp\u003eS\u0026thinsp;=\u0026thinsp;M.L/T S represents the spreadability in grammes.cm/sec, M is the mass in grams, T is the time\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.6.3 \u003cem\u003eIn vitro\u003c/em\u003e Drug Release Studies\u003c/h2\u003e\u003cp\u003e\u003cem\u003eAn in vitro\u003c/em\u003e drug release investigation of TYO-FeONPs gels (F1 to F5) were performed in the dialysis bag approach at 37\u0026deg;C in PBS with a pH of 6.8. The dialysis bag contained all formulation gels and were dialysed in a shaking incubator containing 40 ml of phosphate buffer solution at room temperature. After that, an aliquot (3 ml) was taken at predetermined intervals. As soon as the PBS was taken out, enough fresh PBS was injected to maintain the sink's functionality \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. The amount of TYO released from the TYO-FeONPs gel was measured at a wavelength of 260 nm and conducted in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.6.4 Antimicrobial activity assay\u003c/h2\u003e\u003cp\u003eBacterial culture (P. aeruginosa, MTCC-3541) and B. subtilis (MTCC 1133) were purchased from the Microbial Type Culture Collection and Gene Bank (MTCC) in Chandigarh. The zone inhibition method was used to assay the antibacterial activity following the Kirby-Bauer method \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. The MHA plates were inoculated by spreading 100 \u0026micro;l of P. aeruginosa, B. subtilis, and bacterial culture (the inoculum was made by adjusting 0.5 McFarland units\u0026mdash;approximately 1.5 x 108 CFU/mL from Mueller-Hilton broth). The discs with 10 \u0026micro;l of various concentrations (0 to 100 mg/ml) were then placed on top of the inoculum. The positive control was the ciprofloxacin disc (10 \u0026micro;g) in each plate, while the vehicle control was one disc supplied with solvent only. The plates of P. aeruginosa and B. subtilis were incubated (Basil Scientific Corp. India\u0026mdash;Incubator) for 24 hrs at 37\u0026deg;C. A clear zone created around the disc was checked and noted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.6.5 Time-Kill Kinetics Assay\u003c/h2\u003e\u003cp\u003eThe TYO-FeONPs gel time-kill kinetics were performed using the method outlined by \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.. Concentrations that were three times the TYO-FeONPs gel's MIC were created. Sterile water was transferred to bacterial broth, whereas the negative control was test samples. For twenty-four hours, the bacterial cells' vitality was continually observed. Samples were serially diluted in PBS after being collected at predetermined intervals. At 37\u0026deg;C, they were incubated for the entire night. Bacterial growth in the colonies on the plates was measured after the incubation period. We plotted the graph against the time after calculating and converting the colony-forming unit (CFU) to log CFU/mL.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.6.6 MTT Assay\u003c/h2\u003e\u003cp\u003eAs per the procedure described by [39, 42], the cytotoxicity of samples on the HaCaT cell line (procured from NCCS Pune) was carried out by MTT assay. 10% foetal bovine serum (FBS) was added to the DMEM (Dulbecco's modified Eagle's medium), which was the medium used to cultivate the cells. The cells were kept in an incubator with 5% carbon dioxide and humidity at 37\u0026deg;C. Cell survival was measured using an MTT assay.\u003c/p\u003e\u003cp\u003eThe experiment involved 100 \u0026micro;L of suspension, which contained 1 \u0026times; 104 cancer cells in 96-well plates, and cells were treated with different concentrations of TYO-FeONPs for a further 24 hours. 5 mg/mL of working MTT solution (20 \u0026micro;L) was added to each well and incubated for 4 hours. Untreated cells were termed as control, whereas cells not treated with MTT were blank. We used an Elisa plate reader (iMark, Biorad, USA) to note the absorbance at 540nm \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003e% \u0026#119810;\u0026#119838;\u0026#119845;\u0026#119845; \u0026#119855;\u0026#119842;\u0026#119834;\u0026#119835;\u0026#119842;\u0026#119845;\u0026#119842;\u0026#119853;\u0026#119858;= \u0026#119808;540 \u0026#119847;\u0026#119846; \u0026#119853;\u0026#119851;\u0026#119838;\u0026#119834;\u0026#119853;\u0026#119838;\u0026#119837; \u0026#119836;ells/A540 nm untreated cells X 100\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e2.6.7 Stability Study\u003c/h2\u003e\u003cp\u003eThrough short-term changes in physical stability, we measured the impact of temperature on the TYO-FeONPs gel and kept it for 0, 15, and 30 days at ambient temperature as per the procedure suggested by ICH guidelines. We examined the samples for physicochemical characteristics, such as pH, %DEE, colour, and particle size, at each interval \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Essential Oil Composition\u003c/h2\u003e\u003cp\u003eGC/MS was used to determine the chemical composition of the essential oil. The results of this analysis are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and resulted in the identification of eighty-four bioactive constituents, which shows 100% of total oil. 2,4-Bis(diazo)adamantane, which accounted for 43.67% of the chemicals discovered, was determined to be the most prominent agent, followed by ρ-Methyl salicylate (10.10), p-Cymene-2,5-diol (13.51), Caryophyllene (6.71), and Camphor (7.25). In addition to this, other terpenes are also present in small amounts, such as Borneol, Camphene, p-Cymen-7-ol, etc\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e..\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Characterisation of thyme-loaded iron oxide nanoparticles\u003c/h2\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 FTIR:\u003c/h2\u003e\u003cp\u003eFigure 3a and b shows the FTIR spectra of the thyme oil and thyme-loaded iron oxide nanoparticles (TYO-FeONPs). The O\u0026ndash;H stretch caused the absorption peak in the pure thyme oil at 3239 cm⁻\u0026sup1;. The aliphatic C\u0026ndash;H stretch explains the sharp peak at 2332 cm.=O carbonyl group is responsible for the peak at 1635 cm⁻\u0026sup1;; further evidence of an ester component comes from the C\u0026ndash;O stretch bend seen at 1247 cm⁻\u0026sup1;\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. The C\u0026ndash;H deformations are reflected in the peaks at 787, 1103, and 1247 cm⁻\u0026sup1;. The formulation's absorption peaks, displayed in fig. b, did not significantly differ.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 Particle size and shape of TYO-FeONPs\u003c/h2\u003e\u003cp\u003eThe FE-SEM image of TYO-FeONPs is displayed in Fig.\u0026nbsp;4a and b. The particles in the TYO-FeONPs are dispersed in clusters of various forms, as demonstrated by the microscopic FE-SEM image. Given that it was in great agreement with previously published results, which explain that the population was homogeneous, it is possible to conclude that the optimal particle size of the formulation TYO-FeONPs was satisfactory. The elemental composition of metal nanoparticles is detected using energy dispersive spectroscopy (EDS), which also separates the unique X-rays of various elements into an energy spectrum. The EDS spectra displayed in Fig.\u0026nbsp;4c corroborate the existence of the Fe and O elements. The use of thyme oil in the production process serves as a capping agent around the nanoparticles and provides stability, so the carbon element exists.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3 Percentage Drug Entrapment Efficiency of Thyme-loaded Nanoparticles\u003c/h2\u003e\u003cp\u003eAs per encapsulation efficiency measurement, TYO-FeONPs had an encapsulation effectiveness of 76.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.127% and oil was highly encapsulated. The saturation of the polymer dispersion caused the encapsulation efficiency to reach its maximum and thus remain constant.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Characterisation of TYO-FeONPs Gel\u003c/h2\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1. Physical Parameters:\u003c/h2\u003e\u003cp\u003eThe physical parameters of the prepared formulation and nanoparticles is shown in table no 1. Iron oxide nanoparticle gel were found to be dark black colour with fine consistent and odourless whereas thyme oil loaded iron oxide nanoparticle gel was yellowish brown in colour with greasiness and aromatic odour.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePhysical Parameters\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConsistency\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGreasiness\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOdour\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDark brown /Black\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSmooth powder\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOdorless\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eThyme oil loaded with Fe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eGel\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYellowish Brown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConsistent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSlippery\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAromatic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2 Rheological profile of the TYO-FeONPs topical gel\u003c/h2\u003e\u003cp\u003e\u003cb\u003eThe gel was scrutunized for the pH, viscosity and spreadability and presented in table. 2.\u003c/b\u003e We fabricated a TYO-FeONPs topical gel that may be applied on the skin's surface to achieve consistent dose forms and superior active ingredient carriers. With a pH of 6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 to 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4, the TYO-FeONPs gel is ideal for topical administration and does not irritate the skin and formulation F3 had a pH of 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4. The viscosity was measured at 34,123 cps to cps to 38,142 cps for tested the fabricated gel's spreadability. Between 35,000 and 40,000 cps is the ideal viscosity for topical preparation because it offers spreadability and acceptable flow characteristics and all fabricated formulation shown lower viscosity in comparision to pure thyme oil gel and unveiled shear thining behaviour \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. 9.426\u0026thinsp;\u0026plusmn;\u0026thinsp;2.002 cm was the measured spread distance of the formulation F3. An acceptable spreadability value was attained after determining the gel's viscosity. According to reports in the literature, the topical gel's exceptional viscosity and spreadability make it simple to apply a layer to the skin \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eRheological profile of the TYO-FeONPs topical gel\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSr. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIngredients\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eThyme\u003c/p\u003e\u003cp\u003eOil\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIron oxide\u003c/p\u003e\u003cp\u003eFe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eNanoparticles\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSpreadability\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eviscosity\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003csub\u003e1 blank gel\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.982 cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e36,146 cps\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.876 cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e34,123 cps\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.426\u0026thinsp;\u0026plusmn;\u0026thinsp;2.002 cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e32,201 cps\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e70mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.323 cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e34,142 cps\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e80mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.093 cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e35,221 cps\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e\u003cem\u003e3.3.3 In Vitro\u003c/em\u003e Drug Release Profile of TYO-FeONPs Topical Gel\u003c/h2\u003e\u003cp\u003eIn vitro drug release profile of pure thyme oil (TYO), thyme oil loaded iron oxide nanoparticles (TYO-FeONPs) and thyme oil loaded iron oxide nanoparticles gel (TYO-FeONPs gel) were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e. And buffer solution (pH 6.8) were selected as a medium and amount of drug release were evaluated. As per result, we noted that pure thyme oil gel release 56% of drug within 4 hours as comparison to TYO-FeONPs and TYO-FeONPs gel. We found that 87.05% of drug were released within 8 hrs (thyme oil gel), whereas TYO-FeONPs were released only 78.58% and TYO-FeONPs gel released 75.48% in a buffer solution. The drug release was significantly more in case of TYO-FeONPs gels as compare to other formulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\u003ch2\u003e3.3.4 In Vitro Antimicrobial Activity\u003c/h2\u003e\u003cp\u003eFigure 6a and b and Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003e displayed the antibacterial activity of the TYO-FeONPs, TYO-FeONPs gel and standard drug ciprofloxacin against \u003cem\u003eB.subtilis and P.Aeruginosa\u003c/em\u003e. The topical gel formulations containing thyme oil-loaded iron oxide nanoparticles at a concentration of 60 mg Fe₃O₄ exhibited favourable physicochemical features and superior content relative to alternative formulations. Consequently, these formulations were subsequently chosen for antibacterial investigations. The findings of antimicrobial investigations indicated that the topical gel formulation of thyme oil effectively inhibits microbes during wound healing as reported in the literature\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e][\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. It was observed that the TYO-FeONPs shown higher antibacterial activity than the TYO-FeONPs gel against gram positive (\u003cem\u003eB. subtilis\u003c/em\u003e) and gram negative bacteria \u003cem\u003eP. aeruginosa\u003c/em\u003e the most common resistant strains which is responsible for sepsis, infection and chronic wound\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e][\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. The capability of gels to shown higher antibacterial activity due to various reasons such as interaction between the drug carrier, nano size of formulation, occlusive property\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. The wide range of microbial activity against B. subtilis and P. aeruginosa caused by the encapsulation of iron oxide nanoparticles in the gel (TYO-FeONPs gel) may be the cause. The formulations are promising medicines for treating infected wounds, as evidenced by their antibacterial efficacy.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eZones of inhibition (mm) of TYO-FeONPs and TYO-FeONPs gel against test organisms\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrganism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePositive control (mm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTYO-FeONPs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTYO-FeONPs gel\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eB. subtilis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eP. Aeruginosa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eProposed mechanism for antibacterial activity of TYO-FeONPs and TYO-FeONPs gel\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThrough direct contact with the environment, TYO-FeONPs and TYO-FeONPs gel demonstrated antibacterial efficacy against the test microorganism strains and samples were found to be effective promising antibacterial agent\u003c/p\u003e\u003cp\u003eThe inhibition zone significantly found to be higher when treated with TYO-FeONPs gel topical gel. The proposed mechanism that may be connected to the thyme oil-loaded iron oxide nanoparticles is based on, i.e., iron oxide nanoparticles combat bacteria through oxidative stress by the production of reactive oxygen species and by damaging cell walls and membrane via electrostatic and van der Waals interactions. Bacterial membranes bind metal cations via highly electronegative chemical groups \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Due to their positive charge, metal oxides stick to negatively charged bacteria \u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Electrostatic and van der Waals interactions allow iron oxide nanoparticles to stick to bacterial cell walls and membranes\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Next, iron oxide nanoparticles cluster on the bacterial surface and destroy cell walls and membranes, leaking bacterial material\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e .\u003c/p\u003e\u003cp\u003eDuring fenton reaction there is the ROS production like nonradical hydrogen peroxide, hydroxyl radical and superoxide anion and induced oxidative stress\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. The antibacterial activity by iron nanoparticles inside the cell wall by the release of iron ions and damage the DNA, lipids, and proteins directly, leading to oxidative damage and bacterial death\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe antibacterial activity is depending on bacteria which converts ferric ions to ferrous and, suggesting that ROS generation was their principal antibacterial mechanism \u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. The research found that essential oils and iron oxide nanoparticles work together to kill tested bacterial strains and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\u003ch2\u003e3.3.5. Time-Kill Assays\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003e displays the rates at which TYO-FeONPs gel and TYO-FeONPs killed gram-positive bacteria \u003cem\u003eBacillus subtilis\u003c/em\u003e over the course of 24 hours at 37\u0026deg;C. After a few hours, the result showed that TYO-FeONPs and TYO-FeONPs gel formulation and gel showed a log decrease, indicating a quick bactericidal impact.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\u003ch2\u003e3.3.6. In Vitro Cell Viability Assay\u003c/h2\u003e\u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e9\u003c/span\u003e, HaCaT cell lines were chosen to assess the cytotoxicity of TYO-FeONPs gel. The results showed that after being grown in TYO-FeONPs gels, more than 75% of the cells were still alive. These outcomes demonstrate that the iron oxide loaded thyme oil gel has no negative consequences and that the cells can grow properly. The fact that TYO-FeONPs gel is not harmful to living things and works well with them in these studies shows how useful they could be for in vitro uses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\u003ch2\u003e2.2.6: Stability Study\u003c/h2\u003e\u003cp\u003eDrugs' long-term stability is crucial, yet it can be challenging to attain, particularly in liquid forms. The stability investigation of the TYO-FeONPs gel is displayed in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e4\u003c/span\u003e. TYOs instability due to heat, air, and light was expected to be reduced by encapsulating it in TYO-FeONPs gel. Our study's findings indicate that breakdown and volatility cause a considerable loss of oil components.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStability study of TYO-FeONPs gel at ambient temperature\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDays\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColour\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTexture\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003econsistency\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSmooth\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eConsistent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSmooth\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eConsistent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ewhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003esmooth\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eConsistent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe study successfully synthesised and characterised iron oxide nanoparticles functionalised with thyme oil for dual anticancer and antibacterial purposes. Oil with iron oxide nanoparticles significantly improved its solubility and stability, hence increasing its medicinal potential. The TYO-FeONPs gel showed strong antibacterial efficacy against multiple bacterial strains and showed considerable cytotoxicity towards cancer cells, triggering apoptosis and could be used as a versatile medicine. To make it easier for these nanoparticles to be used in the future, more research needs to be done on them in living things and on how safe and effective they are over time.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are also thankful to the SHUATS, Prayagraj for the continuous support and motivation and thankful to Aakar biotechnology, Lucknow for in vitro anticancer and in vitro antimicrobial study and Panjab University, Bathinda for instrumentation. The author are extremely thankful to biorender (https://www.biorender.com/) for the illustration.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eAS conceptualized, formal analysis and methodology the manuscript; DS review and editing the manscript; AV and DS writing original draft preparation. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003ch2\u003eInstitutional Review Board Statement\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eInformed Consent Statement\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConflicts of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eR. 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Lisitsyn, \u003cem\u003eAntibiotics\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, DOI 10.3390/antibiotics10070884.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"iron oxide nanoparticles, essential oil, thyme oil, anticancer, antibacterial, topical gel","lastPublishedDoi":"10.21203/rs.3.rs-7207816/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7207816/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIron oxide nanoparticles modified with thyme oil (TYO-FeONPs) offer a promising strategy for integrated anticancer and antibacterial treatments. Thyme oil, known for its inherent antibacterial and anticancer characteristics, is frequently constrained by its low water solubility and high volatility. The present study aimed to fabricated and characterized the thyme oil loaded iron oxide nanoparticles topical gel (TYO-FeONPs) and scrutinized its anticancer and antibacterial activity. The physicochemical features of the TYO-FeONPs, were meticulously characterised using FT-IR, FESEM and EDX electron microscopy techniques. GC-MS study exhibited the presence of ρ-Methyl salicylate (10.10), p-Cymene-2,5-diol (13.51), Caryophyllene (6.71), and Camphor (7.25). \u0026nbsp;in the oil. Fe-SEM spectroscopy showed the dispersed the particle of TYO-FeONPs in clusters form with good drug encapsulation of 76.4 ± 3.127%. TYO-FeONPs gel and TYO-FeONPs demonstrated higher drug release and zone of inhibition against \u003cem\u003eP.aerogeniosa\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e . HaCaT cell lines were chosen to assess the cytotoxicity of TYO-FeONPs gel and 75% alive cells. This work emphasises the promise of TYO-FeONPs gel as a dual-purpose therapeutic agent for cancer treatment and bacterial infection control.\u003c/p\u003e","manuscriptTitle":"Iron oxide nanoparticles functionalised thyme oil topical gel for anticancer and antibacterial therapies: fabrication and characterization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 11:58:58","doi":"10.21203/rs.3.rs-7207816/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b8879f92-1416-4929-b0a2-ed2fb7db26b6","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T07:53:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-03 11:58:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7207816","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7207816","identity":"rs-7207816","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}