Document Type : Original Research Article
Subjects
Introduction
The skin, the largest organ in the human body, is a crucial barrier in the immune system, preventing pathogenic microorganisms from invading underlying tissues [1, 2]. However, skin injuries or immune dysfunction can facilitate microbial invasion, leading to infections such as impetigo, cellulitis, and folliculitis, which are particularly common among hospitalized patients [3, 4]. Among bacterial pathogens, Staphylococcus aureus, a gram-positive bacterium, readily colonizes keratinocytes and causes various cutaneous infections, including abscesses, cellulitis, and impetigo [5, 6]. Similarly, Escherichia coli, a gram-negative bacterium, can also contribute to skin and soft tissue infections [7, 8].
Despite the effectiveness of antibiotics in treating bacterial infections, their administration can lead to severe side effects, such as leukopenia, thrombocytopenia, anemia, drug fever, and encephalopathy [9, 10]. Furthermore, the rising incidence of antibiotic resistance, particularly in S. aureus and E. coli, poses a significant global health challenge, with developing countries bearing a disproportionate burden [11, 12]. These limitations have intensified the search for alternative antimicrobial therapies.
Medicinal plants have long been utilized for their therapeutic properties, offering a sustainable source of bioactive compounds with antimicrobial activity [13, 14]. Their affordability, accessibility, and safety make them promising candidates for the treatment of infections. Citrus bergamia (bergamot), a member of the Rutaceae family native to southern Italy, exhibits antimicrobial properties, particularly against gram-negative bacteria [15, 16]. Similarly, Pinus sylvestris (Scots pine), a widely distributed species in Eurasia, demonstrates antibacterial, antiviral, and antifungal effects [17, 18].
Despite their promising antimicrobial properties, essential oils (EOs) face challenges in therapeutic applications due to their volatility and susceptibility to degradation [19]. Incorporating EOs into hydrogel matrices is an effective strategy to modulate their hydrophobicity and enhance stability. Hydrogels are polymeric networks that can disperse drugs within their matrix. Recently, a novel type of hydrogel—nanostructured hydrogels based on nanoemulsions, sometimes called emulgels—has been developed [20, 21]. Nanoemulsions are thermodynamically stable colloidal systems with droplet sizes typically below 200 nm. They offer several advantages in drug delivery, including enhanced solubility of hydrophobic compounds, improved bioavailability, and controlled release properties. Moreover, nanoemulsions enhance the penetration of active ingredients through biological barriers, making them ideal candidates for topical and transdermal applications [22, 23].
When a drug is formulated as a nanoemulsion and subsequently transformed into a gel using a gelling agent such as carboxymethyl cellulose (CMC), the resulting hydrogel benefits from the properties of both systems [24, 25]. CMC is a biocompatible and biodegradable polymer known for its excellent water retention, mechanical stability, and mucoadhesive properties, which enhance the effectiveness of the hydrogel formulation [26, 27]. Consequently, the final nanogel retains the advantages of nanoemulsions—such as improved solubility, stability, and drug delivery—while also offering the benefits of hydrogels, including biocompatibility, biodegradability, and ease of topical application [28-30]. In this study, we developed and characterized nanogels loaded with pine EO, bergamot EO, and their mixture and evaluated their antibacterial activity against E. coli and S. aureus. To the best of our knowledge, this is the first report on the formulation of nanogels containing these specific EOs and the assessment of their antimicrobial potential in such a delivery system.
Escherichia coli (ATCC 25,922) and Staphylococcus aureus (ATCC 25,923) were supplied by the Pasteur Institute of Iran. Carboxymethylcellulose (CMC) and tween 20 were purchased from Merck Chemicals (Germany). The EOs used in this study were purchased from reputable suppliers: Citrus bergamia Risso EO was obtained from Zardband Pharmaceuticals Company (Tehran, Iran), and Pinus sylvestris EO was provided by Dr. Soleimani’s Pharmaceutical Company (Tehran, Iran).
Characterization of bergamot and pine EOs by gas chromatography-mass spectrometry analysis (GC-MS)
Gas chromatographic analysis was conducted using an Agilent 6890 gas chromatograph equipped with a BPX5 capillary column (30 m length, 0.25 mm inner diameter, and 0.25 μm layer thickness). The sample was diluted with n-hexane for component identification and injected (1 µL) into the instrument. The column temperature program was as follows: the initial oven temperature was set at 50 ºC and held for 5 minutes, then increased at a rate of 3 ºC per minute to 240 ºC, followed by a ramp of 15 ºC per minute to 300 ºC, and held for 3 minutes, resulting in a total run time of 75 minutes. The injector temperature was 250 ºC in split mode (1:35), with helium as the carrier gas at a 0.5 ml/min flow rate. The mass spectrometer was an Agilent 5973, operated in electron ionization mode with an ionization voltage of 70 electron volts (eV) and an ion source temperature of 220 ºC. The mass scan range was set between 40 and 500 m/z, and data analysis was performed using ChemStation software. Component identification was achieved by comparing their retention indices with those in reference databases and articles, as well as mass spectra of standard compounds and the computer library.
Preparation and Characterization of nanogels
A fixed amount of pine EO or bergamot EO (100 µL) or a mixture of pine and bergamot EOs (50:50 µL) were mixed with 150 µL of tween 20 at room temperature for 3 minutes. Distilled water was then added dropwise to reach a final volume of 5000 µL, followed by stirring at room temperature for an additional 40 minutes. The particle size and particle size distribution (SPAN) of the nanoemulsions were measured using a dynamic light scattering (DLS) apparatus (K-One Nano Ltd. Korea). The SPAN was calculated using the formula D90 – D10/D50. D10, D50, and D90 represent the particle sizes at which 10%, 50%, and 90% of the particles have diameters more minor than the specified values. Nanoemulsions with particle sizes < 200 nm and SPAN < 1 were considered the optimal sample for the preparation of nanogel [31].
The prepared nanoemulsions containing pine EO, bergamot EO, and their mixture (5000 µL) were gelified by adding 175 mg of CMC and then stirred overnight at ambient temperature to complete the gelation process [31]. The viscosity of nanogels at shear rates of 0.1–100 1/s was analyzed by rheometer apparatus (MCR-302, Anton Paar, Austria). Moreover, proper incorporation of pine and bergamot EOs into nanogel was investigated through Attenuated Total Reflectance Fourier-Transform Infrared (ATR-FTIR) spectroscopy (Tensor II, Bruker, Germany). The spectra of pine EO, bergamot EO, blank gel, and nanogels were examined in the 400-4000 cm− 1 range.
The AATCC100 method was employed to assess the antibacterial properties of the nanogels. Initially, a suspension of the target bacteria was prepared at a standardized concentration of 0.5 McFarland standard (equivalent to 1.5 x 108 CFU/mL). Subsequently, specific quantities of the samples (1000, 500, and 250 milligrams) were added to the bacterial suspension (5 mL). Given that the EO concentration in the nanogel was 10,000 µg/mL, these additions resulted in testing antimicrobial activity at concentrations of 2000, 1000, and 500 µg/mL. After adding the samples, the mixture tubes were incubated at 37°C for 24 hours. Finally, 10 µL of the sample suspension was cultured to Mueller-Hinton agar plates and incubated for 24 hours to determine the growth rate. After the incubation period, the colonies on the plates were counted, and the growth reduction compared to the control group was calculated using the following formula:
Bacterial Growth Percentage = CFU sample / CFU control × 100
Chemical composition of bergamot and pine EOs
The chemical composition of bergamot and pine EOs are listed in Tables 1 and 2, respectively. GC-MS analysis of bergamot EO revealed the identification of 98% of its components, comprising 12 distinct compounds. Among these, the five major compounds were linalool (48.50%), limonene (21.24%), linalool acetate (17.37%), α-terpineol (5.70%), and geranyl acetate (1.74%), which collectively represent the majority of the bergamot EO composition. The constituents of pine EO were fully identified through GC-MS analysis, encompassing seven distinct compounds. Among these, the five major components were α-terpineol (66.69%), ɣ-terpineol (13.10%), terpinen-1-ol (11.15%), cis-β-terpineol (3.00%), and terpinen-4-ol (2.09%), collectively accounting for the majority of the pine EO composition.
The DLS diagrams of nanoemulsions are depicted in Figure 1A-C. The particle size of bergamot EO, pine EO, and their mixture were obtained as 34 ± 4 nm, 10 ± 2 nm, and 42 ± 6 nm, respectively. SPAN of bergamot EO, pine EO, and their mixture were measured at 0.97, 0.99, and 0.97, respectively.
The successful loading of bergamot and pine extract into the nanogel structure is verified by ATR-FTIR spectroscopy. The resulting spectra in the wave number range of 400-4000 cm-1 are illustrated in Figure 2. The spectrum of bergamot EO displays a broad absorption band centered at 3427 cm-1, characteristic of hydrogen-bonded O-H. This suggests the presence of alcoholic compounds in the extract, especially linalool and α-terpineol. Strong absorption bands in the 2980-2865 cm-1 region result from stretching vibrations of aliphatic hydrocarbons. Weaker bands at 3090 and 2743 cm-1 indicate the presence of olefinic and aldehydic C-H groups, respectively. The intense band at 1739 cm-1 indicates the presence of carbonyl compounds, including linalool acetate and geranyl acetate. Additionally, bands at 1645, 1448, and 1372 cm-1 can be assigned to C=C stretching, C-O-H bending, and CH3/CH2 bending vibrations, respectively. The 1245 and 831 cm-1 regions exhibit characteristic bands associated with C-O stretching and out-of-plan (oop) C-H bending vibrations of alkenes and aromatic structures.
The spectrum of pine EO exhibits a strong and broad band centered at 3333 cm-1, suggesting the presence of alcoholic compounds within the extract, especially α-terpineol, ɣ-terpineol, terpinen-1-ol, cis-β-terpineol, terpinen-4-ol, borneol, and fenchol. The C-H and C=C stretching vibrations are observed at 2970-2858 and 1657 cm-1, respectively. Bending vibrations attributed to C-O-H, CH2, and CH3 groups are detected at 1452, 1417, 1376, and 1327 cm-1. Intense and sharp absorption bands in the 1140-989 cm-1 region are assigned to C-O stretching. The other bands are 927-802 cm-1, resulting from =C-H bending vibrations (oop).
The characteristic absorption bands of CMC are observed at approximately 3331 cm-1, corresponding to O-H stretching vibrations. Peaks at 2923 and 1595 cm-1 are attributed to C-H stretching and COO-asymmetric stretching vibrations, respectively. Additionally, 1418 and 1325 cm-1 peaks are associated with CH2 bending, COO-symmetric stretching, and CH3 bending vibrations, respectively. A prominent and sharp absorption band at 1017 cm-1 is assigned to the C-O-C stretching vibrations, characteristic of the glycosidic linkage.
The spectrum of blank gel provides valuable insights into the chemical composition of this nanocarrier. The broad band centered at 3496 cm-1 corresponds to the stretching vibrations of O-H groups from CMC and polyoxymethylene chain of tween 20. Medium peaks at 3070-2908 cm-1 result from C-H bonds’ stretching vibrations. The weak peak at 1744 cm-1 is characteristic of the ester carbonyl group in tween 20. A strong peak of around 1583 cm-1 corresponds to the carboxylate groups (COO-) present in the CMC. Medium absorption at 1431 and 1325 cm-1 are probably due to CH2 bending, COO-symmetric stretching, and CH3 bending vibrations, respectively. Moreover, a strong peak at 1032 cm-1 is associated with the C-O-C stretching vibrations in the ester and ether linkages in tween 20 and CMC.
The bergamot nanogel spectrum exhibits characteristic bands corresponding to both nanogel and bergamot EO, confirming the successful encapsulation of bergamot EO within the nanogel structure. The characteristic peaks at 3526, 2925, 1737, 1583, 1454-1329, 1250, 1058-1033, and 924 cm-1 are attributed to O-H, C-H, C=O, COO-, and C=C stretching, C-O-H and CH2/ CH3 bending, C-O stretching, and C-H bending vibrations (oop), respectively. A significant increase in C-H absorption and the appearance of a carbonyl peak at 1737 cm-1 suggests the interaction between the nanogel matrix and the encapsulated extract components.
The spectrum of pine nanogel indicates the typical absorptions of O-H, C-H, COO-, C=C, and C-O bonds at 3483, 3090-2876, 1587, 1456, and 1042 cm-1, respectively. Specific peaks’ intensity, shape, and position suggest potential nanogel and pine extract interactions. The red shift of the O-H stretching vibration peak indicates the formation of intermolecular hydrogen bonds between the nanogel and the extract components. Enhanced C-H and C-O stretching vibration bands in pine nanogel compared to the blank nanogel confirm the successful loading of pine EO into the nanogel matrix. Moreover, the peaks observed at 932 and 835 cm-1 are attributed to the extract components’ =C-H bending vibrations (oop).
Spectral analysis of the bergamot-pine (mix) nanogel sample identifies characteristic peaks corresponding both extracts and nanogel matrix at 3479 (O-H), 3079- 2927 (C-H), 1734 (C=O), 1589 (COO- and C=C), 1451-1244 (C-O-H, CH2 and CH3), 1045 (C-O), and 920 (oop C-H) cm-1. The observed shift of the hydroxyl group to a lower frequency suggests the formation of hydrogen bonds between the nanogel and the components of both extracts. The position and relative intensity of C-H and C=O signals, being lower than those in bergamot nanogel and higher than those in pine nanogel, indicate the presence of components from both extracts within the biopolymer matrix. The shape of the peaks at 1451-1321 cm-1 and the characteristic peak at 1244 cm-1 provide evidence of both extracts within the matrix. The intense absorption band observed at 1045 cm-1 corresponds to C-O stretching vibrations from pine and bergamot components, tween 20 and CMC.
The viscosity profiles of the nanogels are illustrated in Figure 3A-D. Their viscosity at various shear rates is fully fitted to the Carreau-Yasuda model as the well-known regression of non-Newtonian fluids, where the viscosity decreases with a growing shear rate.
The antibacterial effect of nanogels against E. coli and S. aureus at concentrations of 500, 1000, and 2000 µg/mL are shown in Figures 4 and 5. The control group (untreated bacteria) showed 100% growth across all concentrations, confirming normal bacterial proliferation. The blank gel had minimal antibacterial activity, reducing bacterial growth by only 1%– ~10%, indicating that the gel itself did not significantly contribute to bacterial inhibition. For E. coli, pine nanogel demonstrated the most potent antibacterial effect, reducing bacterial growth to 5% at 2000 µg/mL. Bergamot nanogel showed moderate efficacy, decreasing E. coli growth to 20% at the highest concentration. The mix nanogel exhibited potent antibacterial activity, reducing bacterial growth to 15% at 2000 µg/mL. For S. aureus, pine nanogel was the most effective, reducing bacterial growth to 5% at 2000 µg/mL, while bergamot nanogel reduced it to 15%. The mix nanogel displayed a similar inhibitory effect as pine nanogel, with 5% bacterial growth at the highest concentration. Overall, pine nanogel exhibited superior antibacterial activity against E. coli and S. aureus, followed by the mix nanogel and bergamot nanogel. For better understanding by non-expert readers, a real image example of the grown colonies after treatment with nanogels is presented in Figure 6 from the cultured plates.
One of the major threats to human health is antibiotic resistance, primarily from the overuse of antibiotics in humans, animals, and the environment [32]. Plant EOs have emerged as viable alternatives to combat antibiotic-resistant bacteria due to their diverse bioactive compounds, which reduce the likelihood of bacterial adaptation and resistance [33-35]. Nanoformulation of herbal EOs has emerged as a promising approach to enhance their bioavailability, physicochemical properties, high loading capacity, solubility, stability, and targeted drug delivery [36-38]. Therefore, the present study aimed to develop nanogels incorporating bergamot and pine EOs and their mixture and evaluate their antibacterial efficacy and rheological properties.
The antibacterial evaluation of the developed nanogels demonstrated significant efficacy against E. coli and S. aureus. Pine nanogel exhibited the highest antibacterial activity among the tested formulations, reducing E. coli and S. aureus growth to 5% at 2000 µg/mL. Bergamot nanogel showed moderate inhibition, while the mix nanogel demonstrated strong antibacterial effects comparable to pine nanogel. The antibacterial assessment was conducted using the AATCC100 method, a widely accepted standard for evaluating antimicrobial textiles and surfaces. Unlike disk diffusion, which only measures the inhibition zone, AATCC100 quantifies bacterial survival post-treatment, providing a more dynamic assessment of antimicrobial efficacy. Additionally, while MIC and MBC determine bacterial inhibition at specific concentrations, AATCC100 allows for evaluating bacterial reduction over time or across varying concentrations. It is beneficial for assessing sustained antimicrobial efficacy in formulations like nanogels [39, 40].
Several studies have reported the potent antibacterial effects of nanoformulated EOs against S. aureus and E. coli. For example, treatment with polycaprolactone nanofibers containing Mentha piperita EO reduced the survival rate of S. aureus and E. coli by approximately 50% after 24 hours [41]. Additionally, a nanogel containing 1250 µg/mL of Mentha piperita EO reduced S. aureus growth by 30% and E. coli growth by 95% [42]. Furthermore, caraway nanogel exhibited MIC and MBC values of 0.78 mg/mL and 3.125 mg/mL, respectively, against S. aureus and E. coli [43]. In another study, treatment with 2500 and 5000 µg/mL of nanogel containing Mentha longifolia EO resulted in 3% and 100% growth inhibition of S. aureus, respectively [44]. Notably, camphor nanogel (2500 µg/mL) completely inhibited the growth of both S. aureus and E. coli, and thymol nanogel at the same concentration exhibited 100% inhibition against S. aureus [45]. These findings align with the current study, reinforcing the potential of nanoformulated EOs in antibacterial applications.
The viscosity analysis demonstrated that the nanogels exhibit non-Newtonian, pseudoplastic behavior, as their flow characteristics conform to the Carreau-Yasuda model. This model effectively describes the shear-thinning behavior of polymer-based gels by accounting for viscosity variations across different shear rates [46, 47]. The observed shear-thinning properties are advantageous in biomedical applications, particularly in topical and injectable drug delivery systems, where high viscosity at rest prevents premature drug release. In contrast, lower viscosity under shear facilitates ease of application or injection. Precise rheological characterization aids in optimizing nanogel formulations for targeted delivery, adhesion, and controlled drug diffusion [48, 49].
Over the years, researchers have extensively investigated the mechanism of action of EOs and their major components. It is widely accepted that the antimicrobial effects of EOs are attributed to their ability to disrupt microbial enzymatic systems involved in energy production and cell membrane synthesis [50, 51]. Pinus sylvestris exhibits antibacterial properties against pathogens such as Neisseria gonorrhoeae and Streptococcus suis due to its bioactive components, including α-pinene, β-pinene, and D-limonene. Current evidence suggests that the antibacterial mechanism of P. sylvestris is linked to its ability to induce severe cell membrane disruption and biofilm dispersion [52, 53]. Likewise, Citrus bergamia EO exerts its antimicrobial activity through the lytic effects of its components on microbial cell membranes. This bactericidal activity of Citrus bergamia is primarily attributed to its high limonene content [54, 55].
This study successfully formulated nanogels containing bergamot EO, pine EO, and their mixture. Pine nanogel exhibited the most potent antibacterial effect against E. coli and S. aureus, outperforming the other formulations. With its simple preparation and potent antibacterial properties, pine nanogel is a promising candidate for further research in antimicrobial applications.
Ethics approval and consent to participate
This study has been ethically approved (IR.FUMS.REC.1403.040), and since this research did not involve human study, the informed consent form was thus not used.
Not applicable.
Not applicable
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Not applicable.
Fasa University of Medical Sciences supported this study grant No. 403031.
FD prepared nanoparticles. EA drafted MS in cooperation with MO. RH interpreted ATR-FTIR spectra. ER performed antibacterial assays. ZM revised the MS. MO designed the study, analyzed data, and drafted the manuscript. All authors contributed to drafting and approving the final manuscript.
