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OPEN Nanoencapsulation enhances

stability, release behavior, and
antimicrobial properties of Sage
and Thyme essential oils
Maryam Fakhariha1, Amir Abbas Rafati1, Amir Daraei Garmakhany2 & Azam Zolfaghari Asl1
The increasing demand for natural bioactive compounds in agriculture, food preservation, and
pharmaceuticals has highlighted the need for effective delivery systems to enhance their stability and
bioavailability. In this study, we address this challenge by developing and characterizing silica hollow
nanospheres (HNSs) and hollow polymer nanocapsules (HPNs) for the encapsulation of essential oils
(EOs), specifically those derived from Thyme (Thymus vulgaris) and Sage (Salvia officinalis). The HNSs
were synthesized using tetraethyl orthosilicate (TEOS) via a sol-gel process, while urea-formaldehyde
HPNs (UF–HPNs) were fabricated through in-situ polymerization. The qualitative encapsulation
efficiency, structural integrity, and release behavior of the EOs were analyzed using Fourier transform
infrared spectroscopy (FT–IR), field emission scanning electron microscopy (FE–SEM), and dynamic
light scattering (DLS). The results demonstrated that HNSs, particularly those synthesized via in-situ
techniques, exhibited superior size uniformity, higher oil loading capacity (4.18 mg/g), and controlled
release performance over 102 days. Adsorption studies revealed that HNSs provided higher adsorption
capabilities for Thyme EO, aligning with the Freundlich and Temkin isotherm models. Antimicrobial
studies revealed that encapsulated Thyme EO exhibited strong antibacterial activity, with MIC values
of 4 µL/mL against Escherichia coli (E. coli) and 2 µL/mL against Staphylococcus aureus (S. aureus),
while Sage EO required higher concentrations, with MIC values of 8 µL/mL and 4 µL/mL, respectively.
Notably, the encapsulation of Thyme EO in HNSs resulted in enhanced antimicrobial performance
compared to HPNs, likely due to the porous silica matrix allowing for sustained EO release. The
encapsulated EOs also modulated peroxidase enzyme activity, further supporting their potential for
biological applications. These findings suggest that HNS-based encapsulation offers a robust and
sustainable approach for enhancing the efficacy of natural antimicrobial agents, making them suitable
for industrial applications in biopesticides, food safety, and therapeutic formulations.

Keywords Essential oils, Nano-encapsulation, Silica Hollow nanospheres, Polymer nanocapsules,

Antimicrobial activity, Controlled release

Nanotechnology has significantly transformed the agricultural, pharmaceutical, and food industries by offering
innovative strategies to enhance the stability, bioavailability, and controlled release of bioactive compounds.
Essential oils (EOs), derived from various plant species, have gained considerable attention due to their
antimicrobial, antioxidant, and therapeutic properties. However, their high volatility and susceptibility to
environmental degradation limit their direct industrial applications. To address these challenges, nanocapsule-
based encapsulation techniques have emerged as a promising approach to protect EOs from environmental
factors while enabling controlled and sustained release, thereby enhancing their functional performance1–8.
Among various nanocarriers, silica hollow nanospheres (HNSs) and urea-formaldehyde hollow polymer
nanocapsules (UF–HPNs) have demonstrated exceptional potential for EO encapsulation. HNSs, synthesized
via a sol-gel process using tetraethyl orthosilicate (TEOS), are characterized by their robust structure, high
surface area, and tunable porosity, making them suitable for encapsulating and releasing bioactive compounds
efficiently9,10. The hydrolysis and condensation of TEOS under alkaline conditions form a Si–O–Si network,
ensuring the structural integrity of the nanocapsules. Additionally, surfactants such as cetyltrimethylammonium

1Department of Physical Chemistry, Faculty of Chemistry and Petroleum Sciences, Bu-Ali Sina University, P.O.Box
65174, Hamedan, Iran. 2Department of Food Science and Technology, Faculty of Engineering and Natural Resources
of Toyserkan, Bu-Ali Sina University, Hamedan, Iran. email: [email protected]

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bromide (CTAB) can be employed to control the size and morphology of HNSs, contributing to their enhanced
stability and uniformity11–15. Such properties are particularly valuable in applications requiring precise particle
size control, such as drug delivery and biopesticide formulations16.
On the other hand, UF–HPNs, synthesized through in-situ polymerization of urea and formaldehyde, provide
a highly cross-linked polymeric matrix that forms a durable protective shell around volatile compounds17–19.
This polymeric network significantly improves the mechanical stability of the nanocapsules, allowing them to
withstand environmental stresses and ensuring prolonged release of the encapsulated compounds. The ability of
UF–HPNs to create rigid and protective shells makes them ideal candidates for agricultural, pharmaceutical, and
food-related applications, where sustained release and enhanced stability are essential20,21.
A critical aspect of evaluating these nanocapsules is the comprehensive characterization of their structural
and functional properties. Techniques such as Fourier transform infrared spectroscopy (FT–IR), field
emission scanning electron microscopy (FE–SEM), and dynamic light scattering (DLS) play a crucial role in
assessing qualitative encapsulation efficiency, size distribution, surface morphology, and stability in aqueous
environments22,23. FT–IR is widely used to confirm the successful encapsulation of EOs by identifying
characteristic functional groups19, while SEM provides insights into the nanocapsule surface morphology24.
Furthermore, DLS enables the determination of hydrodynamic diameter and polydispersity index (PDI), which
are key parameters for assessing the stability and dispersibility of nanocapsules in different media21.
Beyond their structural properties, encapsulated EOs exhibit biological activity, particularly in modulating
enzyme activity and demonstrating antimicrobial effects23,25,26. Peroxidase enzyme activity, which plays a vital
role in plant defense mechanisms and food preservation, can be significantly influenced by the presence of
encapsulated EOs27–29. Additionally, the antimicrobial efficacy of these nanocapsules against common foodborne
and pathogenic bacteria such as E. coli and S. aureushas been well documented. Encapsulation enhances the
antimicrobial effectiveness of EOs by protecting their active components from degradation and allowing for
controlled and prolonged release, making them a viable alternative to synthetic preservatives and antibiotics2–5.
This study systematically investigates the synthesis, characterization, and functional performance of HNSs
and UF–HPNs encapsulating Sage (Salvia officinalis) and Thyme (Thymus vulgaris) essential oils. The research
focuses on assessing qualitative encapsulation efficiency, release kinetics, adsorption behavior, and antimicrobial
efficacy, with an emphasis on potential applications in controlled-release biopesticidal formulations, food
preservation, and pharmaceutical applications. By utilizing advanced analytical techniques and exploring
the biological activity of encapsulated EOs, this study provides valuable insights into the factors governing
the effectiveness of nanocapsules as delivery systems, contributing to the development of more efficient and
sustainable strategies for EO utilization across various industries1–5.
This study offers a novel perspective by concurrently investigating three critical aspects—stability, release
behavior, and antimicrobial efficacy—of essential oils encapsulated in both silica and polymer nanocarriers.
Unlike previous works that often focus on a single type of oil or encapsulation method, this research uniquely
compares both Sage and Thyme essential oils, employing dual synthesis approaches (in-situ and immersion
methods). This comparative and integrative analysis provides new insight into the optimization of encapsulation
strategies for multifunctional applications, which has been overlooked in the current literature.

Materials and methods

Chemicals and reagents
All chemicals and reagents used in this study were of analytical grade and purchased from reputable suppliers.
Cetyl trimethyl ammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), sodium dodecyl sulfate (SDS),
ammonia solution, urea, ammonium chloride, resorcinol, octanol, formaldehyde solution (37%), ethanol,
guaiacol, hydrogen peroxide, monosodium phosphate, disodium phosphate, sodium hydroxide, phosphoric
acid, and dimethyl sulfoxide (DMSO) were obtained from Merck, Darmstadt, Germany. BHI broth and BHI
agar were sourced from Condalab, Madrid, Spain, and the Sage and Thyme EOs were acquired from Lavendor
Co., Tehran, Iran.

Synthesis of HPN and HNS containing EOs

In-situ polymerization
HPNs containing Sage and ThymeEOs were synthesized using an in-situ polymerization technique. Briefly, 130
mL of distilled water was mixed with 5 mL of 5% (w/w) SDS in a 500 mL beaker at room temperature and stirred
at 200 rpm. Urea (2.5 g), ammonium chloride (0.25 g), and resorcinol (0.25 g) were added, and the mixture was
allowed to stabilize for 10 min. The pH was adjusted to 3.5 using 1% (v/v) hydrochloric acid. Subsequently, 1.00
mL of the selected EO and 1–2 drops of octanol (as an antifoaming agent) were introduced into the mixture.
The resulting emulsion was homogenized by ultrasonication for 10 min. Following this, 6.335 g of 37% (w/w)
formaldehyde was added, and the mixture was sonicated for an additional 4 h. The formed nanocapsules were
separated by centrifugation at 13,000 rpm, thoroughly washed with distilled water, and freeze-dried for storage30.

Immersion method
For the immersion method, hollow silica and HPNs were first synthesized as described in the in-situ
polymerization method but without the addition of EOs. Once dried, the nanocapsules were immersed in EOs
at a polymer-to-oil ratio of 1:4 and a silica-to-oil ratio of 1:5.5. The immersion process was carried out for 24 h to
allow for adequate adsorption of EOs. After immersion, the excess oil was removed via centrifugation, and the
capsules were rinsed with distilled water and freeze-dried30.

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Sol-gel method for HNSs

HNSs encapsulating EOs were synthesized using the sol-gel technique. In this process, a solution of 100.00
mL double-distilled water and 0.082 g CTAB was sonicated for 5 min. While stirring, 0.50 mL of EO and 1.00
mL of 25% ammonia were added to the alkaline medium, followed by 1.00 mL of TEOS. The reaction mixture
was subjected to sonication for 2 h. The resulting nanocapsules were collected by centrifugation at 13,000 rpm,
washed with distilled water, and freeze-dried for further use31.

Characterization of nanocapsules
The synthesized nanocapsules were characterized using FE–SEM (TESCAN–MIRA3 Czech) to examine the
surface morphology of the nanocapsules. However, DLS was utilized to measure the particle size distribution
of the nanocapsules. On the other hand, FTIR spectra were recorded using a Spectrum 65 FT–IR device
(Perkin Elmer, Switzerland) to identify the chemical functionalities present in the nanocapsules. The UV–Vis
spectroscopic analysis was conducted using SPEKOL 2000/1 spectrophotometer (Analytik Jena, Germany) to
determine the optical properties and quantify the encapsulated EOs.

Adsorption and release studies

Adsorption thermodynamics
To investigate the adsorption behavior of EOs onto the synthesized nanocapsules, 0.03 g of adsorbent (polymer
or silica) was added to a 10.00 mL flask containing 5.00 mL of EO solutions at varying concentrations. The
solutions were incubated at 25 °C under continuous stirring at 150 rpm for 24 h. After incubation, the mixtures
were filtered using syringe filters, and the concentration of the remaining EO in the solution was determined by
measuring absorbance at the maximum wavelength (λmax) using a UV–Vis spectrophotometer.

Release rate studies

To evaluate the release behavior of the encapsulated EOs over time, 0.1 g of oil-loaded nanocapsules were
weighed and stored at room temperature, shielded from moisture. The capsules were periodically weighed at set
intervals, and the retention of EOs was calculated based on the weight loss over time, until stabilization of the
weight indicated the retention capacity.

Enzyme inactivation and antibacterial activity

Peroxidase activity assay
The peroxidase activity in cabbage and apple extracts was measured using guaiacol and hydrogen peroxide
as substrates. In a 1 cm path-length cuvette, 2.87 mL of the guaiacol-hydrogen peroxide mixture, 0.10 mL of
the enzyme extract, and 0.03 mL of EO were combined. The reaction progress was monitored by measuring
absorbance at 470 nm, with reduced peroxidase activity in the presence of EO compared to a control sample32.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The MIC and MBC of Thyme and Sage EOs against E. coli and S. aureus were determined using the broth
microdilution method. Serial dilutions of the EOs (from 512 µL/mL to 1 µL/mL) were prepared in a 96-well
microplate. Bacterial suspensions at a concentration of 106CFU/mL were added to each well. The plates were
incubated at 37 °C for 18–24 h. The MIC was defined as the lowest concentration of EO that inhibited visible
bacterial growth. The MBC was established by subculturing samples from wells showing no bacterial growth
onto Mueller–Hinton Agar (MHA) and incubating at 37 °C for 24 h. The lowest concentration that resulted in
no bacterial growth was recorded as the MBC33.

Results and discussion

Synthesis mechanism and characteristics of nanocapsules
Synthesis of HNS
The synthesis of HNSs utilized TEOS, a compound chosen for its well-established hydrolysis and condensation
properties, both of which are crucial for forming a stable silica network. The process begins in an alkaline
medium, facilitated by ammonia, which initiates the hydrolysis of TEOS. During hydrolysis, the OR groups
in TEOS are replaced by OH groups, producing Si(OH)₄ and alcohol. This preparation step is vital for the
subsequent condensation phase. In the condensation reaction, Si(OH)₄ molecules interact, forming Si–O–Si
bonds, with water as a byproduct. The reactions involved are as follows:

1. Hydrolysis of alkoxysilanes

Si(OR)4 + 4H2O → Si(OH)4 + 4ROH.

2. Condensation reaction

(OR)3Si–OH + HO–Si(OR)3 → (OR)3Si–O–Si(OR)3 + H2O.

The cationic surfactant, CTAB, plays an essential role in the process by forming micelles in the aqueous medium.
These micelles, characterized by their positively charged surfaces, attract the negatively charged hydrolyzed and
condensed silica species, leading to the deposition of a silica shell around the micelles. This interaction is critical
for controlling the size and shape of the nanocapsules. The positively charged CTAB micelles serve as a template

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for silica deposition, ensuring the nanocapsules maintain a uniform spherical structure, which is important for
applications requiring consistency in particle size and morphology.
The success of this synthesis method is evident in the uniformity of the produced nanocapsules. Precise
control over the hydrolysis and condensation reactions, combined with CTAB’s templating role, ensures that the
HNSs exhibit structural integrity. This stability is especially valuable for controlled-release systems and other
applications where consistent performance is critical.

Mechanism of UF–HPN synthesis

The synthesis of UF–HPNs follows a polymerization process where urea reacts with formaldehyde, resulting
in the formation of hydroxymethyl urea derivatives. These derivatives undergo further polymerization and
cross-linking to form a stable polymer network. The process consists of three key stages: (i) formation of
hydroxymethyl derivatives: urea reacts with formaldehyde to produce hydroxymethyl urea derivatives, which
act as intermediates during the polymerization process. (ii) Polymerization: the hydroxymethyl derivatives
further react, forming long polymer chains. This stage constructs the fundamental polymer framework that
will encapsulate the EOs. (iii) Cross-Linking: the polymer chains undergo cross-linking, creating a three-
dimensional network. This cross-linking step is crucial for providing mechanical stability to the nanocapsules,
allowing them to withstand environmental variations without degrading.
Scheme 1illustrates the reaction pathway for the UF polymer synthesis, showing the formation of
hydroxymethyl derivatives, the subsequent polymerization, and the critical cross-linking phase that imparts
stability and functionality to the nanocapsules34.
UF was selected as the encapsulating material due to its ability to form a rigid and durable polymer shell,
particularly suited for protecting volatile compounds such as EOs. The cross-linked structure not only ensures
mechanical durability but also acts as a barrier, regulating the release of the encapsulated material. This makes
the nanocapsules ideal for applications requiring a controlled and extended-release profile.

Adsorbent characterizations
FT–IR spectroscopy provides critical insights into the chemical structure and qualitative encapsulation
efficiency by identifying key functional groups within the nanocapsule matrices. The spectral data confirmed the
successful encapsulation of Sage and Thyme EOs in both HNS and UF–HPNs, retaining the chemical integrity
of the oils and nanomaterials. The FT–IR spectrum of HNS (Fig. 1a) exhibited characteristic absorption peaks
between 1000 and 1130 cm−1, corresponding to the asymmetric stretching vibrations of Si–O–Si bonds, a
critical structural component of the silica network. The peak at 963 cm−1 is attributed to Si–OH stretching,
confirming the presence of surface silanol groups, while the broad band around 3228 cm−1, associated with O–H
stretching vibrations, indicates hydroxyl groups. These findings are consistent with previous studies confirming
the structural integrity of nanosilica frameworks for adsorptive applications. The FT–IR spectra of nanosilica
encapsulating Thyme and Sage oils (Fig. 1b and c) displayed similar peaks for Si–O–Si bonds, confirming that
the encapsulation process did not disrupt the silica network. Additionally, new peaks were observed in the
2961–2925 cm−1 range, corresponding to C–H stretching vibrations, which are indicative of the presence of
aliphatic hydrocarbons from the EOs. These peaks, rather than those corresponding to C = C bonds, suggest
that the encapsulation successfully retained the organic compounds from the EOs. This observation confirms
that the encapsulation was successful and that the stability of both the EOs and the silica matrix was maintained
throughout the process.
The FTfIR spectra for UF–HPNs (Fig. 2a) revealed characteristic bands around 1636 cm⁻¹ for C = O stretching
and 1563 cm⁻¹ for N–H bending, typical of secondary amide bonds, confirming the successful formation of the
polymer matrix. In the presence of Sage and Thyme oils, peaks in the 2960–2925 cm⁻¹ range showed slight shifts,
indicating interactions between the polymer matrix and the EOs (Fig. 2b and c). These shifts suggest hydrogen

Scheme 1. The synthesis and cross-linking of urea-formaldehyde polymers under both alkaline and acidic
conditions34.

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Fig. 1. FT–IR of (a) HNS; (b) HNS–Thyme–EO; (c) HNS–Sage–EO.

bonding between the oils and the polymer, as supported by previous studies highlighting such interactions in
polymer-based encapsulation systems. The FT–IR results confirmed theation of EOs within both silica and
HPNs, with no significant disruption of the nanocapsule matrix. This preservation of chemical structure is
crucial for maintaining the functionality and controlled release properties of the encapsulated oils. The FTIR
spectra of pure essential oils are also given in the Supplementary material in Figure S1.
SEM images revealed that HNSs exhibit a spherical morphology with smooth surfaces, as shown in Fig. 3a.
The uniform size distribution, ranging from 55.0 nm to 84.2 nm for Sage oil encapsulation (Fig. 3b and d)
and 43.2 nm to 66.7 nm for Thyme oil encapsulation (Fig. 3c and e), confirms successful formation and
encapsulation. These findings align with previous studies that highlight the ability of HNSs to provide high
surface area and uniform morphology, which are crucial for enhancing encapsulation efficiency and retention
of volatile compounds such as essential oils (EOs). Similar morphological characteristics have been reported
in silica-based nanocarriers, where their well-defined spherical structure contributes to improved stability and
controlled release properties35.
The SEM image of UF–HPNs (Fig. 3f) also displayed a spherical morphology, consistent with previous
observations of polymer-based nanocapsules used for controlled release applications. The particle size of UF–
HPNs encapsulating Thyme oil (Fig. 3g and i) ranged from 140.5 nm to 175.2 nm, while those encapsulating
Sage oil (Fig. 3h and j) ranged from 118.7 nm to 267.0 nm. The smooth, crack-free surfaces indicated effective
encapsulation without aggregation, a feature commonly observed in polymeric nanocapsules designed for
sustained release of bioactive compounds. Studies have demonstrated that polymer-based nanocarriers typically
exhibit a more compact and uniform structure compared to inorganic matrices, contributing to their enhanced
stability and controlled diffusion profiles36.
The morphological consistency and smooth surfaces observed in both nanocapsule systems further confirm
the efficiency of the encapsulation process, qualitatively. The absence of surface deformations or structural

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Fig. 2. FT–IR of (a) HPNs (b) HPNs–Thyme–EO (c) HPNs–Sage–EO.

Fig. 3. SEM images of (a) HNS, (b) HNS–Sage–EO (immersed) (c) HNS–Thyme–EO (immersed), (d)
HNS–Sage–EO (in-situ), (e) HNS–Thyme–EO (in-situ), (f) HPNs, (g) HPNs–Thyme–EO (immersed), (h)
HPNs–Sage–EO (immersed), (i) HPNs–Thyme–EO (in-situ), (j) HPNs–Sage–EO (in-situ).

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disruptions suggests that these nanocapsules are well-suited for applications where stability, protection, and
prolonged release of encapsulated materials are critical. These observations are in agreement with prior research
on nanocapsule systems used for drug delivery and agricultural formulations, where particle uniformity and
integrity play key roles in ensuring optimal performance37.
Dynamic Light Scattering (DLS) measurements were conducted to evaluate the hydrodynamic size
(Z-average) and polydispersity index (PDI) of the encapsulated essential oil samples in different formulations
and preparation methods. The results, summarized in Table 1, provide insights into the size distribution and
uniformity of the nanocapsules, which are critical parameters for their performance in various applications.
The hydrodynamic size (Z-average) of the nanocapsules varied significantly based on the type of essential
oil (Thyme or Sage), encapsulation method (immersed or in-situ), and capsule matrix (HNS or HPN). Thyme-
immersed HNS nanocapsules exhibited the smallest hydrodynamic size (233.80 ± 11.7 nm) with a moderate
PDI of 0.26, indicating a relatively narrow size distribution. Sage-immersed HNS nanocapsules showed a larger
Z-average of 320.48 ± 16.0 nm and a higher PDI (0.30), suggesting greater heterogeneity in size.
In the in-situ encapsulation method, the Z-average for both Thyme and Sage formulations increased
significantly, with Thyme reaching 423.67 ± 21.2 nm and Sage measuring 324.85 ± 16.2 nm. The PDI values
remained moderate (0.26 and 0.19, respectively), indicating a controlled particle size distribution despite the
increase in size.
On the other hand, Thyme and Sage-immersed HPN nanocapsules demonstrated much larger hydrodynamic
sizes (608.22 ± 30.4 nm and 672.92 ± 33.6 nm, respectively) compared to their HNS counterparts. These samples
also exhibited extremely low PDI values (0.05 and 0.02), signifying highly uniform particle size distributions.
The in-situ method further increased the hydrodynamic size for both Thyme and Sage HPN samples, with
Thyme reaching 688.00 ± 34.4 nm and Sage extending to 794.23 ± 39.7 nm. While the PDI values remained low
for Sage (0.07), the Thyme sample showed a slight increase to 0.19, suggesting a broader size distribution in the
in-situ formulation.
The differences in Z-average values can be attributed to several factors: (i) EO Type: The molecular
composition and polarity of Thyme and Sage EOs influence their interactions with the nanocapsule matrix,
potentially affecting swelling and aggregation behavior; (ii) Encapsulation method: In-situ encapsulation may
lead to better dispersion and uniformity, whereas post-synthesis immersion could result in partial aggregation
or surface adsorption, thereby altering the hydrodynamic size; (iii) Capsule matrix: The structural differences
between HNS and HPNs, including porosity, surface charge, and rigidity, impact their interaction with the
surrounding medium, leading to variations in hydration layers and size distribution.
This analysis underscores the importance of tailoring encapsulation parameters to optimize the performance
of essential oil-loaded nanocapsules for specific applications.

Release test results

The release profiles of EOs encapsulated within nanocapsules were monitored over a 102–day period to evaluate
their potential for controlled release applications (Fig. 4; Table 2). Controlled release is a key factor in determining
the suitability of these nanocapsules for various industrial, agricultural, and biomedical uses, as it directly affects
the delivery efficiency and duration of the bioactive compounds.
HNSs demonstrated faster and higher release rates than polymer-based nanocapsules. This difference can be
attributed to the porous structure of the silica matrix, which enables rapid diffusion of the encapsulated EOs.
Among the different HNSs, those produced via in-situ methods exhibited the most rapid release of EOs due to
their larger size and increased loading capacity. The porous structure of the silica provides numerous diffusion
pathways for EOs, enhancing their release rate.
Several studies support these findings, highlighting that the large surface area and porosity of HNSs
significantly enhance their release efficiency. This is particularly beneficial for applications requiring rapid
diffusion of active compounds, such as in agricultural biopesticides, where a quick response to pest outbreaks
is essential. In such cases, the fast release of EOs from HNSs ensures a swift and effective action against pests.
Conversely, HPNs exhibited a slower, more controlled release profile compared to their silica counterparts.
This slower release is due to the denser, less porous nature of the polymer matrix, which hinders the diffusion
of the encapsulated EOs. The more uniform and stable encapsulation environment within the polymer matrix
forms an effective barrier to diffusion, thus providing sustained release over an extended period.

Sample Z average (nm) PDI

HNS, Thyme Immersed 233.80 ± 11.7 0.26
HNS, Sage Immersed 320.48 ± 16.0 0.30
HNS, Thyme In-Situ 423.67 ± 21.2 0.26
HNS, Sage In-Situ 324.85 ± 16.2 0.19
HPN, Thyme Immersed 608.22 ± 30.4 0.05
HPN, Sage Immersed 672.92 ± 33.6 0.02
HPN, Thyme In-Situ 688.00 ± 34.4 0.19
HPN, Sage In-Situ 794.23 ± 39.7 0.07

Table 1. DLS results showing hydrodynamic diameter (Z-average) and PDI of nanocapsules containing EOs.

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Fig. 4. Release profile of essential oils from silica and polymer nanocapsules over 102 days, comparing
immersed and in-situ methods for Thyme and Sage oils.

This observation aligns with other studies that demonstrate the effectiveness of HPNs in providing a
prolonged and steady release, making them ideal for applications such as drug delivery systems and long-term
agricultural treatments. The lower polydispersity index (PDI) of HPNs indicates their higher stability, which
enhances their ability to deliver a controlled and consistent release over time.
Across all types of nanocapsules, Thyme EO exhibited a higher release rate compared to Sage EO. This
difference can be explained by the intrinsic properties of the oils, such as molecular weight, volatility, and
interaction with the encapsulating matrix. Thyme EO, with its lower molecular weight and higher volatility,
diffuses more readily through both silica and polymer matrices, resulting in a faster release profile.
On the other hand, Sage EO, characterized by a higher molecular weight and lower volatility, has a stronger
interaction with the encapsulating matrices, leading to a slower release.
Sage essential oil (EO), which contains a higher proportion of sesquiterpenes and diterpenes compared to
ThymeEO, exhibits lower volatility due to the increased molecular weight of these components. Studies have
shown that essential oils with larger molecular structures tend to have lower vapor pressures, reducing their
tendency to evaporate rapidly38. This characteristic leads to stronger interactions with encapsulating matrices,
as larger and less volatile molecules exhibit greater affinity for polymeric and inorganic hosts through hydrogen
bonding and van der Waals interactions39. Consequently, encapsulated SageEO demonstrated a slower release
rate, aligning with previous reports indicating that oils with higher molecular weights diffuse more slowly
through nanoscale carriers due to steric hindrance and matrix retention effects40. The controlled release of
Sage EO observed in this study is consistent with these established principles, reinforcing the importance of
molecular weight and volatility in determining release kinetics.
Similar results have been reported in other research, where differences in EO composition significantly
influence their release kinetics when encapsulated in nanomaterials. Adjusting the release pattern of EOs and
the encapsulation methods allows for optimizing the delivery system to suit the specific requirements of the
intended application.

Equilibrium adsorption analysis of EOs

Using Eq. 1, the equilibrium adsorption capacity (qe) was calculated from the experimental data for Thyme and
Sage EOs adsorbed onto the synthesized hollow nanocapsules.
C0 − Ce
qe = V (1)
m

where qe (mg/g) is the amount of EO adsorbed on the HNS or HPN at considered equilibrium concentration, C0
(mg/L) is the initial concentration of the EO in solution, Ce (mg/L) is the equilibrium concentration of the EO in
solution, V (L) is the volume of the EO solution and m (g) is the mass of adsorbent used.
The equilibrium data were analyzed in accordance with the Langmuir, Frundlich and Temkin sorption
isotherm models. A nonlinear regression analysis was applied to evaluate the adsorption parameters for all
isotherms. All of the models are listed in Table 3.

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Released time period of EO

Material name Absorbed elasticity percentage Day 1 Day 3 Day 5 Day 10 Day 14 Day 19 Day 24 Day 32 Day 40 Day 54 Day 67 Day 77 Day 102

| https://doi.org/10.1038/s41598-025-00022-5
0% 0.5% 0.6% 0.8% 1.2% 1.5% 1.6% 1.7% 1.8% 1.8% 1.8% 1.9% 2.1%
Sage/HPN (Immersed) 14.00%
(± 0.05) (± 0.05) (± 0.05) (± 0.07) (± 0.11) (± 0.11) (± 0.12) (± 0.13) (± 0.11) (± 0.13) (± 0.11) (± 0.10) (± 0.12)
0% 1.3% 2.1% 3.1% 4.5% 5.4% 5.6% 5.7% 6.3% 6.3% 6.4% 6.5% 7.1%
Sage/HNS (Immersed) 18.75%
(± 0.05) (± 0.11) (± 0.12) (± 0.14) (± 0.15) (± 0.21) (± 0.22) (± 0.21) (± 0.27) (± 0.28) (± 0.25) (± 0.23) (± 0.31)
0% 0.2% 0.5% 0.7% 1.1% 1.5% 1.6% 1.7% 1.8% 1.8% 1.8% 1.9% 2.1%
Thyme/HPN (Immersed) 11.00%
(± 0.05) (± 0.05) (± 0.05) (± 0.06) (± 0.11) (± 0.12) (± 0.12) (± 0.12) (± 0.13) (± 0.12) (± 0.14) (± 0.13) (± 0.17)
0% 1.5% 2.7% 4.2% 5.4% 5.5% 6.9% 7.1% 7.9% 8.0% 8.2% 8.5% 9.5%
Thyme/HNS (Immersed) 18.00%
(± 0.05) (± 0.11) (± 0.13) (± 0.14) (± 0.13) (± 0.21) (± 0.30) (± 0.30) (± 0.32) (± 0.30) (± 0.35) (± 0.31) (± 0.35)
0% 1.7% 2.8% 3.9% 4.7% 4.8% 4.9% 5.0% 5.4% 5.4% 5.5% 5.7% 6.2%
Sage/HPN (In Situ) 19.80%
(± 0.05) (± 0.11) (± 0.12) (± 0.14) (± 0.18) (± 0.16) (± 0.24) (± 0.24) (± 0.25) (± 0.21) (± 0.23) (± 0.21) (± 0.29)

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0% 3.1% 4.8% 7.3% 8.7% 9.4% 9.5% 9.7% 10.1% 10.2% 10.3% 10.6% 11.8%
Sage/HNS (In Situ) 19.70%
(± 0.05) (± 0.16) (± 0.14) (± 0.32) (± 0.39) (± 0.35) (± 0.35) (± 0.40) (± 0.40) (± 0.41) (± 0.38) (± 0.40) (± 0.39)
0% 0.3% 0.9% 1.5% 2.3% 2.3% 2.3% 2.4% 2.7% 2.7% 2.9% 2.9% 3.2%
Thyme/HPN (In Situ) 10.30%
(± 0.05) (± 0.06) (± 0.08) (± 0.12) (± 0.14) (± 0.17) (± 0.13) (± 0.14) (± 0.14) (± 0.13) (± 0.15) (± 0.12) (± 0.17)
0% 3.3% 5.2% 8.6% 10.5% 11.7% 12.3% 12.8% 13.9% 13.9% 14.3% 14.9% 15.8%
Thyme/HNS (In Situ) 22.80%
(± 0.05) (± 0.14) (± 0.17) (± 0.38) (± 0.39) (± 0.40) (± 0.40) (± 0.40) (± 0.40) (± 0.41) (± 0.40) (± 0.41) (± 0.42)

Table 2. Comparison of the percentage of loaded EO with the percentage of released EO over 102 days.

9
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Isotherm Equation Linear Form Plot

1 1
qe = = +
qm KL Ce Ce
Langmuir 1+KL Ce qe KL qm qm Ce Ce
qe vs.Ce
1
Freundlich qe = KF Ce1/n lnq e = lnK F + n lnCe
lnq e vs. lnCe
Temkin qe = RT
b ln(K T Ce ) qe = RT
b lnK T + RT
b lnCe qe vs. lnCe

Table 3. Isotherm models and their linear forms.

Fig. 5. Equilibrium adsorption isotherm of (a) Thyme–EO on HNS; (b) Thyme–EO on HPNs; (c) Sage–EO on
HNS; (d) Sage–EO HPNs, at 25 °C.

The adsorption equilibrium behavior of both EOs on HNS and HPNs was assessed to evaluate their
effectiveness in encapsulating EOs for controlled release applications.
The equilibrium adsorption data for Thyme EO on HNSs at 25 °C are detailed in Table S1 (in Supplementary
material), while Fig. 5a shows the relationship between the equilibrium concentration (Ce) and the adsorbed
amount (qe). The adsorption capacity (qe) of Thyme EO on HNSs increased with the initial concentration of the
EO, reaching a maximum of 5.32 mg/g at a Ce of 128.06 ppm. The adsorption isotherm showed Langmuir-like
behavior, implying monolayer adsorption on a homogeneous surface. This high adsorption capacity is attributed
to the porous structure and large surface area of the HNSs, which provide numerous active sites for adsorption.
Similarly, the equilibrium adsorption of Thyme EO on HPNs at 25 °C (Fig. 5b) followed the same increasing
trend, although the maximum adsorption capacity reached only 3.63 mg/g at a Ce of 98.18 ppm. This lower
capacity compared to HNSs is likely due to the denser structure of the polymer matrix, which limits the number
of active sites for adsorption. Despite this, the HPNs exhibited sufficient adsorption capacity for applications
requiring controlled release.
HNSs showed a higher adsorption capacity than HPNs at lower concentrations. At an initial concentration
of 24.00 ppm, HNSs achieved a qe of 0.61 mg/g, while HPNs only reached 0.19 mg/g. The porous structure of
silica facilitates more efficient uptake of Thyme EO, especially at low concentrations, making it better suited
for applications needing rapid adsorption. As the concentration of Thyme EO increased, HNSs continued to
outperform HPNs. At a Ce of 94.89 ppm, HNSs achieved an adsorption capacity of 4.18 mg/g, whereas HPNs
reached 3.63 mg/g. This difference in performance is attributed to the porous nature of the silica, which offers
faster and more efficient adsorption.

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A similar trend was observed for the adsorption of Sage essential oil onto HNS (Fig. 5c), while the adsorption
behavior of Sage EO on HPNs (Fig. 5d) displayed significant data scatter, preventing clear trends. This
inconsistency may stem from weaker interactions between Sage EO and the nanocapsule surfaces, potentially
due to the molecular structure of Sage oil or other factors not explored in this study.
At higher concentrations, both HNS and HPNs converged to the same maximum adsorption capacity of
5.32 mg/g at a Ce of 128.06 ppm. This suggests that, at elevated concentrations, both types of nanocapsules
saturate their adsorption sites and reach similar performance levels. Despite silica’s superior performance at
lower concentrations, both materials demonstrated comparable adsorption capacities at maximum loading. The
porous structure of HNSs provides a larger surface area and more active sites for adsorption, contributing to
their higher adsorption capacity, especially at lower concentrations. This makes HNSs ideal for applications
requiring rapid and high-capacity adsorption. The denser structure of HPNs slows the adsorption process by
limiting diffusion, but this also makes them better suited for applications needing a sustained and controlled
release over time.
The adsorption of Thyme EO onto both HNS and HPNs was further evaluated using three isotherm models:
Langmuir, Freundlich, and Temkin. The parameters for each model are presented in Table S2 (in Supplementary
material), with the corresponding adsorption isotherms shown in Fig. 6a and b. The Langmuir isotherm assumes
monolayer adsorption on a finite number of homogeneous adsorption sites. The qm values for HNS and HPN
were − 20.01 mg/g and − 8.88 mg/g, respectively, indicating a higher adsorption capacity for HNS. This aligns

Fig. 6. Thyme–EO adsorption isotherms on (a) HNS adsorbent, (b) HPNs adsorbent.

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with the porous structure of silica, which offers more active sites. However, the negative KL values suggest the
need for further investigation into the adsorbate-adsorbent interactions.
The KF parameter, which reflects the adsorption capacity, was determined to be 0.016 for HNS-based
nanocapsules and 0.0075 for HPN-based nanocapsules. This result suggests that HNS-based nanocapsules have
a higher adsorption capacity compared to HPN-based nanocapsules, indicating that the HNS matrix provides
more favorable surface properties for adsorption.
The AT values of the Temkin model for HNS and HPNs were 0.048 and 0.0412, respectively, indicating weak
adsorbent-adsorbate interactions for both materials. The slightly higher AT value for HNS implies stronger
interactions than in HPNs, corresponding with HNS’s higher adsorption capacity.
The values of R2 and RMSE as two common error functions are listed in Table S3 (in Supplementary material).
The values of RMSE about Freundlich model for Thyme were smallest and the value of R2 was greatest, among
the three isotherms.
In summary, HNSs exhibited superior adsorption performance due to their larger surface area and porous
structure, making them ideal for rapid and high-capacity adsorption. HPNs, while displaying lower initial
adsorption, showed more controlled and sustained release, advantageous for applications requiring prolonged
efficacy. These findings highlight the need to select appropriate materials based on the desired application,
whether it be for rapid adsorption or controlled release.

Kinetic studies
The adsorption kinetics of Thyme and Sage EOs on synthesized hollow nanocapsules were studied by calculating
the amount of oil adsorbed over time (qt) using Eq. 2:
C0 − Ct
qt = V (2)
m

Experimental data are displayed in Table S4 (in Supplementary material) and Fig. 7a and d, revealing the time-
dependent adsorption of EOs on both HNS and HPN. The adsorption kinetics of Thyme EO on HNSs, depicted
in Fig. 7a, show a steady increase in adsorption rate over time until equilibrium is reached. The kinetic data
for Thyme EO on HPNs (Fig. 7b) suggest a slightly different adsorption pattern, likely due to variations in the

Fig. 7. Kinetic adsorption of (a) Thyme–EO on HNS, (b) Thyme–EO on HPNs, (c) Sage–EO on HNS, (d)
Sage–EO on HPNs at 25 °C.

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polymer structure affecting the surface properties and adsorption behavior. For Sage EO, the adsorption kinetics
on HNSs are presented in Fig. 7c. This shows a slower adsorption process compared to Thyme oil, which may
be attributed to differences in the molecular properties of Sage oil and its interaction with the silica surface.
Sage EO adsorption on HPNs (Fig. 7d) also shows slower kinetics, indicating sustained adsorption over time.
These findings highlight that both the type of nanocapsule material and the EO used significantly influence the
adsorption rate. HNSs tend to have a faster adsorption process for Thyme EO, while HPNs offer a more controlled
release. Sage EO generally adsorbs more slowly than Thyme oil across both nanocapsule types, suggesting that
different formulations could be tailored for specific applications requiring different release rates.
The adsorption kinetics of EOs on HNS and HPNs were further analyzed using various kinetic models (PFO,
PSO, Elovich, and intra-particle diffusion) via nonlinear regression methods. All of the models are listed in
Table S5 (in Supplementary material) and the values of parameters obtained by different kinetic models are
summarized in Tables S6 to S9 (in Supplementary material), reveal that the pseudo-second-order model provides
the best fit for Thyme oil on HNSs (Fig. 8a), while the Elovich model offers a better fit for HPNs (Fig. 8b).
For Sage EO adsorption on HNSs (Fig. 8c), the pseudo-second-order model again provides the best fit, while
the intra-particle diffusion model gives a more accurate representation for adsorption on HPNs (Fig. 8d). These
variations in model fitting underscore the complexity of the adsorption process, which may involve multiple
mechanisms.
The values of different errors are listed in Table 4. For Thyme EO adsorption kinetics on HNS, the values of
error functions about pseudo-second order kinetic model were smallest among the five kinetics models. The
obtained data show that the pseudo-second order kinetic model explains the adsorption in a better way. On
the other hand, examination of the kinetic error functions of the adsorption of Thyme on HPN shows that its
adsorption follows the Elovich equation. For Sage EO adsorption kinetics, the intra-particle diffusion model
shows better error functions for both HNS and HPN adsorbents.

Fig. 8. Typical fitting data with various kinetic models for adsorption of (a) Thyme–EO on HNS, (b) Thyme–
EO on HPNs, (c) Sage–EO on HNS, (d) Sage–EO on HPNs at 25 °C.

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HNS HPN
EO Error function MOE Intra-particle diffusion Elovich PSO PFO MOE Intra-particle diffusion Elovich PSO PFO
R² 0.921 0.865 0.934 0.938 0.906 0.819 0.836 0.882 0.858 0.770
Thyme
RMSE 0.305 0.244 0.174 0.168 0.207 0.231 0.238 0.202 0.224 0.282
R² 0.846 0.895 - 0.882 0.861 0.914 0.927 - 0.926 0.905
Sage
RMSE 3.006 2.292 - 2.430 2.643 2.893 2.467 - 2.490 2.810

Table 4. Error function values for nonlinear fitting of kinetic adsorption data of EOs on nanocapsules.

Fig. 9. (a) Enzyme activity curve of white cabbage peroxidase under the influence of Thyme and Sage essential
oils; (b) Enzyme activity curve of apple peroxidase under the influence of Thyme and Sage essential oils.

Enzyme peroxidase inactivation

The impact of encapsulated EOs on peroxidase enzyme activity was studied to explore their potential applications
in biological systems. The enzyme peroxidase plays a crucial role in plant defense mechanisms, food processing,
and preservation. Figure 9a and b illustrate the effects of Sage and Thyme EOs on the activity of peroxidase in
white cabbage and apple trees.

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MIC MBC
Bacteria Thyme/HPN Sage/HPN Thyme/HNS Sage/HNS Thyme/HPN Sage/HPN Thyme/HNS Sage/HNS
E. coli - - 1 µL/mL 4 µL/mL - - 2 µL/mL -
S. aureus 8 µL/mL 16 µL/mL 1 µL/mL 2 µL/mL 16 µL/mL - 1 µL/mL 16 µL/mL

Table 5. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of
encapsulated EO for E. coli and S. aureus bacteria.

Sage EO significantly enhanced the activity of peroxidase in white cabbage, as indicated by the steeper slope
of the absorption curve compared to the control. This suggests that Sage oil may act as an activator of peroxidase,
improving the enzyme’s catalytic efficiency. The increase in enzyme activity could be attributed to specific
interactions between the components of Sage oil and the enzyme, possibly leading to conformational changes
that enhance its performance. This enhancement of peroxidase activity could be beneficial in agriculture,
where increased enzyme activity might bolster plant defenses against environmental stressors and pathogens.
Conversely, in food processing, where peroxidase activity influences oxidation reactions, the increase in enzyme
activity might require careful management to avoid potential negative effects on product quality, such as
discoloration or nutrient degradation.
Thyme EO exhibited a biphasic effect on peroxidase activity. Initially, the oil appeared to slow enzyme activity,
as shown by the lower slope of the absorption curve compared to the control. However, over time, the enzyme
activity increased, eventually surpassing that of the control. This complex response suggests an initial inhibitory
interaction, likely due to Thyme oil components binding to the enzyme’s active site, temporarily reducing catalytic
efficiency. As the interaction continues, either conformational changes or the removal of inhibitory components
may lead to enhanced enzyme activity. Understanding this biphasic response is critical for applications in which
precise control over enzymatic activity is essential, such as food preservation, where prolonged enzymatic
activity may need to be regulated.
In apple trees, both Sage and Thyme EOs significantly increased peroxidase activity, indicating their potential
as natural activators of plant defense mechanisms. This enhancement in peroxidase activity suggests that these
EOs could be used in agriculture as natural elicitors to stimulate a plant’s defense response, potentially reducing
the need for synthetic pesticides. However, the observed effects on enzyme activity also raise questions about
the broader physiological impacts of EOs on plant growth, development, and fruit quality, all of which need to
be further explored.
The study of peroxidase inactivation by encapsulated EOs provides valuable insights into their potential use in
agriculture and food processing. The ability of EOs to modulate enzyme activity—either enhancing or inhibiting
it—has far-reaching implications for these industries. These findings suggest that careful consideration of the
specific EO and its interaction with enzymes is essential for optimizing its use in different applications.

MIC and MBC test results

The antimicrobial efficacy of encapsulated Thyme and Sage EOs was evaluated using MIC and MBC tests against
Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). While EOs are known for their antimicrobial
properties, encapsulation enhances their stability and allows for controlled release, thereby improving their
effectiveness. The MIC and MBC values obtained for both EOs are presented in Table 5, highlighting their
potential as natural antimicrobial agents in various applications.
Thyme EO demonstrated potent antimicrobial activity, with MIC values of 4 µL/mL against E. coli and 2 µL/
mL against S. aureus, and MBC values of 4 µL/mL for both bacteria. The high antimicrobial efficacy of Thyme
oil is primarily attributed to its phenolic compounds, particularly thymol and carvacrol, which are known to
disrupt bacterial cell membranes and interfere with essential cellular processes. Encapsulation of Thyme EO in
nanocapsules further improved its antimicrobial performance by protecting it from degradation and enabling
controlled release, maintaining sustained antibacterial activity over time. The low MIC and MBC values
suggest that encapsulated Thyme EO could serve as an effective alternative to synthetic antibiotics, especially in
applications where natural and sustainable antimicrobial agents are desired.
Sage EO exhibited antimicrobial activity, although it required higher concentrations than Thyme oil. The
MIC values were 8 µL/mL for E. coli and 4 µL/mL for S. aureus, with MBC values of 16 µL/mL and 8 µL/mL,
respectively. The lower antimicrobial efficacy of Sage EO compared to Thyme oil is likely due to differences in their
chemical compositions. Sage oil contains a mix of terpenes, which, while possessing antimicrobial properties, are
generally less potent than the phenolic compounds found in Thyme oil. Nevertheless, the encapsulated Sage oil
still demonstrated significant antibacterial activity, making it a viable option for applications requiring a broader
spectrum of activity.
Encapsulation of Thyme EO in HNSs showed superior antimicrobial performance compared to HPNs. The
porous structure of the silica matrix allows for a slow, sustained release of the EO, maintaining its antimicrobial
activity over an extended period. This sustained release is particularly advantageous in applications requiring
long-term antibacterial effects, such as food preservation, medical coatings, and surface disinfection. The
superior antimicrobial activity of Thyme oil encapsulated in silica emphasizes the importance of selecting
appropriate encapsulation materials to enhance the functional properties of EOs.
Although HPNs also provided controlled release, their performance in enhancing the antimicrobial efficacy
of encapsulated EOs was less pronounced than that of HNSs. This difference can be attributed to the structural
properties of the polymers used, which may affect the release rate and stability of the EO. However, HPNs still

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offer potential advantages, such as biocompatibility, which could be beneficial in specific applications, including
medical or pharmaceutical uses.
The MIC and MBC test results highlight the strong antimicrobial potential of encapsulated Thyme EO,
particularly when incorporated into HNSs. These findings suggest that encapsulated EOs could serve as highly
effective antimicrobial agents for a variety of industries, ranging from food preservation to healthcare, where
natural and sustainable solutions are increasingly in demand.

Conclusion
This study successfully demonstrated the nano-encapsulation of Sage (Salvia officinalis) and Thyme (Thymus
vulgaris) essential oils using HNS and urea-formaldehyde hybrid polymeric nanocapsules (UF–HPNs). The
encapsulation significantly improved the stability, controlled release, and antimicrobial efficacy of the essential
oils. Structural and morphological characterization confirmed the integrity of the nanocapsules, while biological
evaluations highlighted their potential as effective biopesticidal agents.
The release test results demonstrate that the choice of encapsulating material and method significantly
influences the release kinetics of essential oils (EOs). Hollow nanosilica (HNS), with its porous structure,
facilitates a faster release rate, making it suitable for applications requiring immediate and potent release, such as
emergency agricultural treatments or rapid drug delivery. In contrast, hybrid polymeric nanocapsules (HPNs),
offering a more controlled and prolonged release, are ideal for sustained-release applications, including drug
delivery systems and long-term agricultural biopesticides.
The ability to tailor the release profile of EOs by selecting different nanocapsule materials and encapsulation
methods offers a versatile platform for applications in pharmaceuticals, food preservation, and cosmetics, where
controlled and prolonged release is essential. The selection of biopolymer-based nanocapsules provides a cost-
effective approach due to their availability and relatively low production costs. Additionally, the emulsion-based
encapsulation techniques used in this study are scalable and compatible with industrial production, making
them commercially viable for large-scale applications. To further advance this field, future research should focus
on:

• Investigating the effect of synthesis conditions, such as polymerization time, precursor ratios, and surfactant
concentrations, on encapsulation efficiency and release kinetics.
• Assessing the efficacy of encapsulated EOs against a broader range of microorganisms, including fungal and
viral pathogens, to explore new applications in food safety and medicine.
• Conducting large-scale agricultural trials to evaluate the effectiveness of nano-encapsulated EOs under real
environmental conditions, ensuring their practicality as sustainable biopesticides.
• Exploring the use of biodegradable polymers, metal-organic frameworks (MOFs), or lipid-based carriers to
enhance controlled release and bioavailability.
• Investigating the co-encapsulation of multiple bioactive compounds to enhance antimicrobial, antioxidant,
and pesticidal properties through synergistic interactions.
• Assessing the long-term environmental impact and potential toxicity of encapsulated EOs to ensure their
safety for widespread application.

Data availability
All data supporting the findings of this study are available from the corresponding author upon reasonable
request.

Received: 11 February 2025; Accepted: 24 April 2025

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Scientific Reports | (2025) 15:18373 | https://doi.org/10.1038/s41598-025-00022-5 17

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Acknowledgements
The authors greatly acknowledge Bu-Ali Sina University for the financial support from the Grant Research
Council.

Author contributions
Maryam Fakhariha: Investigation, Methodology, Visualization, Writing-original draft, Data curation, Formal
analysis, Software. Amir Abbas Rafati: Project administration, Conceptualization, Supervision, Funding acqui-
sition, Writing-review & editing, Resource, Validation. Amir Daraei Garmakhany: Advisor, data interpretation,
reviewed and edited the manuscript. Azam Zolfaghari Asl: Investigation, Methodology, Visualization, Writ-
ing-original draft, Software.

Declarations

Conflict of interest
The author declares no conflict of interest.

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