Preparation, Characterization, and Antibiofilm Activity of Free and Nanoencapsulated Tetradenia riparia (Hochst.) Codd Leaf Essential Oil

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please revise the manuscript content suggested in enclosed file

Comments for author File: Comments.docx

Author Response

REVIEWER 1

Comment 

Please revise the following paper content.

Response: This typo has been deleted

 

Response: 

 

 

 

Response

This error has been corrected. The title of Table 1 now correctly reads GC–MS.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The article is a study on the preparation, characterization and antibiofilm effect of Tetradenia riparia (Hochst) Codd leaf essential oil, both free and nanoencapsulated. In general, the article lacks development in all sections. The introduction clearly outlines the study to be conducted, however, its innovative nature is not apparent. It is also necessary to improve the introduction by including examples of very recent references in the field. The methodology section, perfectly described, is consistent with the study being conducted. It leads the authors to numerous results, some of which are simply presented and commented on. However, in my opinion, there is no discussion. This discussion should be clearly presented and would thus better highlight the authors' innovative findings. Section 3.5, In Vitro Release, absolutely must be developed, as this is where the new aspects lie. It is therefore important to expand upon it, perhaps even supplementing it with more advanced experiments (or at least putting them into perspective in the conclusion).

The section on characterizing the essential oil should be simplified as it refers to previously published articles. Alternatively, it should highlight any new findings.

Tables should be positioned within the body of the text when they are explained.

Author Response

REVIEWER 2

REVIEWER

The article is a study on the preparation, characterization and antibiofilm effect of Tetradenia riparia (Hochst) Codd leaf essential oil, both free and nanoencapsulated. In general, the article lacks development in all sections. The introduction clearly outlines the study to be conducted, however, its innovative nature is not apparent. It is also necessary to improve the introduction by including examples of very recent references in the field.

ANSWER

New text and new references have been added to the manuscripr below.

“Biofilm-associated infections with Staphylococcus aureus are difficult to treat even after administration of antibiotics that according to the standard susceptibility assays are effective  (Kaushik et al., 2024, Brandquist and Kielian, 2025). Currently, the assays used in the clinical laboratories to determine the sensitivity of S. aureus towards antibiotics are not representing the behaviour of biofilm-ass.ociated S. aureus, since these assays are performed on planktonic bactéria (Sultan et al., 2022).”

References

Kaushik A, Kest H, Sood M, Steussy BW, Thieman C, Gupta S. Biofilm Producing Methicillin-Resistant Staphylococcus aureus (MRSA) Infections in Humans: Clinical Implications and Management. Pathogens. 2024 Jan 15;13(1):76. doi: 10.3390/pathogens13010076. PMID: 38251383; PMCID: PMC10819455

Brandquist ND, Kielian T. Immune dysfunction during S. aureus biofilm-associated implant infections: opportunities for novel therapeutic strategies. NPJ Biofilms Microbiomes. 2025 Jul 25;11(1):144. doi: 10.1038/s41522-025-00782-y. PMID: 40715142; PMCID: PMC12297603.)

(Sultan AR, Tavakol M, Lemmens-den Toom NA, Croughs PD, Verkaik NJ, Verbon A, van Wamel WJB. Real time monitoring of Staphylococcus aureus biofilm sensitivity towards antibiotics with isothermal microcalorimetry. PLoS One. 2022 Feb 16;17(2):e0260272. doi: 10.1371/journal.pone.0260272. PMID: 35171906; PMCID: PMC8849495.

Hedayati S, Tarahi M, Azizi R, Baeghbali V, Ansarifar E, Hashempur MH. Encapsulation of mint essential oil: Techniques and applications. Adv Colloid Interface Sci. 2023 Nov;321:103023. doi: 10.1016/j.cis.2023.103023. Epub 2023 Oct 12. PMID: 37863014.

 

REVIEWER

The methodology section, perfectly described, is consistent with the study being conducted. It leads the authors to numerous results, some of which are simply presented and commented on.

REVIEWER

Section 3.5, In Vitro Release, absolutely must be developed, as this is where the new aspects lie. It is therefore important to expand upon it, perhaps even supplementing it with more advanced experiments (or at least putting them into perspective in the conclusion).

ANSWER

It has now been included in the text:

1. repiaria essential oil, which contains a higher proportion of sesquiterpenes and diterpenes 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 rapidly ( Singh and Pulikkal, 2022).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 interactions (Gupta et al., 2016). Consequently, encapsulated EO 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 effect (Yammine et al., 2024). The controlled release of EO observed in this study is consistent with these established principles, reinforcing the importance of  molecular weight and volatility in determining release kinetics.

REVIEWER

The section on characterizing the essential oil should be simplified as it refers to previously published articles. Alternatively, it should highlight any new findings.

ANSWER

After submitting the manuscript, I received the following message from the editor: “All research articles submitted to Future Pharmacology that describe natural products should fulfill complete details of any methodology used for isolation and purification;   Structure elucidation of the mixture components (by MS, LC–MS, NMR)...”  

REVIEWER

Tables should be positioned within the body of the text when they are explained.

ANSWER

“The instructions were followed in preparing the manuscript. “Use the Microsoft Word template or LaTeX template to prepare your manuscript. See “Preparing Manuscripts in LaTeX” for further details;”

REVIEWER

However, in my opinion, there is no discussion. This discussion should be clearly presented and would thus better highlight the authors' innovative findings.

ANSWER

After incorporating all the suggestions and revisions indicated by the reviewer, the section results and discussion was revisited

 

Reviewer 3 Report

Comments and Suggestions for Authors

The current manuscript reflects an interesting work on the development of a novel nanoplatform containing Tetradenia riparia (Hochst) Codd leaves essential oil. Several relevant assays have been performed, and the results appear to be adequate, therefore I only advise on the current alterations before acceptance for publication:

- In the abstract, the authors claim that “NP stability was studied using zeta potential. Its value was determined to be 27 around -23.1 mV, which indicates that NP are in fact stable”, but ZP value is not a certainty of NP stability; in order to confirm formulation stability, specific stability assays should be performed; hence, the authors should rewrite this, and say that a high ZP value could mean that the formulation could have high colloidal stability, through electrostatic repulsion; but it should not be concluded that the NP are in fact stable due to the ZP value in itself, but instead NP stability should be based on stability results; the same conclusions should be rewritten on subsection “3.4. Encapsulation Efficiency”, which says “NPs exhibited a zeta 223 potential of −23.1 mV, indicating adequate stability [43].”;

- In the Introduction section, more should be said on polymeric nanoparticles, namely why these NP were selected (instead of other types of nanosystems, such as lipid nanoparticles, liposomes, nanoemulsions, etc.), and additional advantages that EO encapsulation can provide;

- An administration route should be mentioned for the developed formulation: is it intended for intravenous administration? Or another route, such as oral, topical, etc.?;

- Was the method used for NP preparation developed from scratch? Or was it based on previously described and published methods? If it’s this second option, then the appropriate references should be added;

- It is mentioned that “NP size ranged from 221.9 to 396.5 nm.”, but was this the intended size range? It is not enough to mention that the particles are “nanometric”, since target size ranges differ with the intended purpose, for example different administration routes have different target values; this should be further discussed;

- Section “3.4. Encapsulation Efficiency” mentions ZP as well, hence this should be mentioned in the title, or a different subsection should be created for ZP only;

- In vitro drug release results should be further commented, is 25% of release enough? What could be the impact of these results? Also the authors say that these results are “consistent with previous studies”, but don’t mention these previous studies, and should;

- In the “3.6. Antibacterial Activity” section the authors mention that “enhanced antibacterial activity after encapsulation would have been desirable”, but the fact that the EO has a controlled release from the NP makes an enhanced antibacterial activity difficult, at least in assays performed in a short period of time; hence, this comment should be rewritten;

- Figures and Tables should appear right after they are first mentioned in the text.

Author Response

REVIEWER  3

REVIEWER

The current manuscript reflects an interesting work on the development of a novel nanoplatform containing Tetradenia riparia (Hochst) Codd leaves essential oil. Several relevant assays have been performed, and the results appear to be adequate, therefore I only advise on the current alterations before acceptance for publication:

- In the abstract, the authors claim that “NP stability was studied using zeta potential. Its value was determined to be 27 around -23.1 mV, which indicates that NP are in fact stable”, but ZP value is not a certainty of NP stability; in order to confirm formulation stability, specific stability assays should be performed; hence, the authors should rewrite this, and say that a high ZP value could mean that the formulation could have high colloidal stability, through electrostatic repulsion; but it should not be concluded that the NP are in fact stable due to the ZP value in itself, but instead NP stability should be based on stability results; the same conclusions should be rewritten on subsection “  3.4. Encapsulation Efficiency”, which says “NPs exhibited a zeta 223 potential of −23.1 mV, indicating adequate stability [43]

ANSWER

Differential scanning calorimetry (DSC) analysis provided valuable insights into the thermal behavior and stability of the polymeric nanoparticles by quantifying the enthalpy changes associated with melting transitions. The DSC parameters are summarized in Table 2. Blank nanoparticles (NPs) exhibited a melting temperature (Tm) of 54.29 °C and a high melting enthalpy (ΔHm) of 429.63 J/g, reflecting a more ordered crystalline structure of the polymeric matrix.

In contrast, nanoparticles loaded with essential oil (EO) showed a slight reduction in melting temperature (52.71 °C) accompanied by a pronounced decrease in melting enthalpy to 115.83 J/g. This substantial reduction in ΔHm indicates a decrease in polymer crystallinity, suggesting that the incorporation of the EO disrupts the crystalline organization of the PLA matrix.

The DSC thermograms presented in Figure 2 corroborate these findings, as blank NPs displayed a larger and more defined endothermic peak, whereas EO-loaded NPs exhibited a markedly reduced peak area. Such thermal behavior indicates strong interactions between the encapsulated EO and the polymeric chains, leading to a more amorphous structure. This structural modification facilitates polymer melting and enhances molecular mobility, which is favorable for controlled and sustained release of the encapsulated EO.

Overall, the DSC results confirm successful encapsulation and demonstrate that EO incorporation modulates the thermal properties of the nanoparticles, supporting their suitability as controlled-release delivery systems for volatile and thermolabile bioactive compounds.

In addition, as described in Section 2.8, a stability assay was conducted over 30–45 days at 25 ± 2 °C, in accordance with RDC 45 (August 9, 2012) [17]. Antibacterial activity was subsequently evaluated using the broth microdilution method following CLSI guidelines. The MIC values of the nanoparticles remained unchanged throughout the 45-day period, indicating that nanoencapsulation preserves EO stability and prevents oxidative degradation.

REVIEWER

- In the Introduction section, more should be said on polymeric nanoparticles, namely why these NP were selected (instead of other types of nanosystems, such as lipid nanoparticles, liposomes, nanoemulsions, etc.), and additional advantages that EO encapsulation can provide;

ANSWER

It is now stated in the introduction:

Poly(lactic acid) (PLA) polymeric nanoparticles have attracted increasing attention in biomedical and microbiological research due to their biocompatibility, biodegradability, and low toxicity, as they are metabolized into láctica acid, a naturally occurring compound (Makadia and Siegel, 2011; Danhier et al., 2012). In addition, PLA-based nanocarriers efficiently protect labile bioactive compounds, including essential oils and other hydrophobic antimicrobial agents, from volatilization and environmental degradation. These systems also enable controlled release and improved aqueous dispersibility, which enhances biological efficacy and reduces adverse effects, supporting their potential application in antimicrobial and antifungal strategies (Panyam and Labhasetwar, 2003; Kumari et al., 2010).

REVIEWER

- An administration route should be mentioned for the developed formulation: is it intended for intravenous administration? Or another route, such as oral, topical, etc.?;

ANSWER

It is now stated in the text:

The use of nanoparticles in products for topical application improves the permeation/penetration of the bioactive compound in to deeér layers of the skin, providing a depot effect with sustained drug release and specífic cellular and subcellular targeting. Nanocarriers provide advances in dermatology and systemic treatment (Raszewska-Famielec and Flieger, 2022)

 

REVIEWER

Was the method used for NP preparation developed from scratch? Or was it based on previously described and published methods? If it’s this second option, then the appropriate references should be added;

ANSWER

Bibliographic references were included in Materials and Methods

 

REVIEWER

- It is mentioned that “NP size ranged from 221.9 to 396.5 nm.”, but was this the intended size range? It is not enough to mention that the particles are “nanometric”, since target size ranges differ with the intended purpose, for example different administration routes have different target values; this should be further discussed;

ANSWER

As mentioned above the initial focus is on topical use dermatological products.

 

REVIEWER

- Section “3.4. Encapsulation Efficiency” mentions ZP as well, hence this should be mentioned in the title, or a different subsection should be created for ZP only;

-ANSWER

ZP was transferred to the section 3.3. Differential Scanning Calorimetry (DSC)

REVIEWER

In vitro drug release results should be further commented, is 25% of release enough? What could be the impact of these results? Also the authors say that these results are “consistent with previous studies”, but don’t mention these previous studies, and should;

ANSWER

This comment was included in the manuscript:

1. repiaria essential oil, which contains a higher proportion of sesquiterpenes and diterpenes 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 rapidly ( Singh and Pulikkal, 2022).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 interactions (Gupta et al., 2016). Consequently, encapsulated EO 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 effect (Yammine et al., 2024). The controlled release of EO observed in this study is consistent with these established principles, reinforcing the importance of  molecular weight and volatility in determining release kinetics.

REVIEWER

- In the “3.6. Antibacterial Activity” section the authors mention that “enhanced antibacterial activity after encapsulation would have been desirable”, but the fact that the EO has a controlled release from the NP makes an enhanced antibacterial activity difficult, at least in assays performed in a short period of time; hence, this comment should be rewritten;

ASWER

As can be seen in the conclusions, although antibacterial activity against Staphylococcus aureus was not enhanced, nanoencapsulation preserved the EO’s efficacy while significantly reducing cytotoxicity. These findings demonstrate that PLA nanoparticles are an effective delivery system for optimizing the stability and safety of T. riparia EO, reinforcing their suitability for future pharmacological development.

REVIEWER

- Figures and Tables should appear right after they are first mentioned in the text.

ANSWER

“The instructions were followed in preparing the manuscript. “Use the Microsoft Word template or LaTeX template to prepare your manuscript. See “Preparing Manuscripts in LaTeX” for further details;”

 

 

Reviewer 4 Report

Comments and Suggestions for Authors

Comments on manuscript ID futurepharmacol-4053470

The authors of the manuscript “Preparation, Characterization, and Antibiofilm Effect of Free and Nanoencapsulated Tetradenia riparia (Hochst) Codd Leaves Essential Oil” had the goal to prepare polymeric nanoparticles containing T. riparia essential oil, employing nanoprecipitation. The formed nanoparticles were characterised by their physicochemical properties, and their cytotoxicity and antibiofilm activity against S. aureus were assessed.

The English is fine, and the cited literature data are appropriate.

Abstract:

Line 26, please delete  “aure”.

Materials and Methods

Subsection 2.2, please provide the method of relative quantification of the component present and identified in the investigated EO (GC/FID?)

Subsection 2.4, lines 107-109, please, give a brief description of the methods used

Subsection 2.6, line 125, please, explain the chosen absorbance at 325 nm for EO encapsulation efficiency

Subsection 2.7, lines 133,134, please provide more data regarding the quantitative analysis of the in vitro release profile, and the results presented in Figure 3

When first mentioned, the full Latin name of the plant should be provided – species, author name, and family as well. However, afterwards, the short or common name form should be used throughout the text (T. riparia, line 174…please, check through the text and correct where appropriate).

Results and Discussion

Subsection 3.1, lines181-187, please give the data regarding diterpenoids, as they significantly contributed to the chemical profile of the investigated EO

Author Response

REVIEWER 4

Comments on manuscript ID futurepharmacol-4053470

The authors of the manuscript “Preparation, Characterization, and Antibiofilm Effect of Free and Nanoencapsulated Tetradenia riparia (Hochst) Codd Leaves Essential Oil” had the goal to prepare polymeric nanoparticles containing T. riparia essential oil, employing nanoprecipitation. The formed nanoparticles were characterised by their physicochemical properties, and their cytotoxicity and antibiofilm activity against S. aureus were assessed. The English is fine, and the cited literature data are appropriate.

Comment

Abstract:

Line 26, please delete  “aure”.

Answer

Deleted

Reviewer

Materials and Methods

Subsection 2.2, please provide the method of relative quantification of the component present and identified in the investigated EO (GC/FID?)

Answer

The reference was added

Reviewer

Subsection 2.4, lines 107-109, please, give a brief description of the methods used

Answer

The methods were detailed and the references added

Reviewer

Subsection 2.6, line 125, please, explain the chosen absorbance at 325 nm for EO encapsulation efficiency

Answer

UV–visible spectroscopy at 325 nm was employed because essential oil constituents, particularly oxygenated and conjugated terpenoids, exhibit characteristic absorption bands in this region due to π→π and n→π* electronic transitions. This wavelength allows sensitive and selective quantification of the essential oil with minimal interference from the polymeric nanoparticle matrix and has been widely used for determining encapsulation efficiency in essential oil–loaded polymeric nanoparticles*

Reviewer

Subsection 2.7, lines 133,134, please provide more data regarding the quantitative analysis of the in vitro release profile, and the results presented in Figure 3

Answer

The following paragraphs have been added to the current tex

“T. repiaria essential oil, which contains a higher proportion of sesquiterpenes and diterpenes 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 rapidly ( Singh and  Pulikkal, 2022).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 interactions (Gupta et al., 2016). Consequently, encapsulated EO 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 effect (Yammine et al., 2024). The controlled release of EO observed in this study is consistent with these established principles, reinforcing the importance of  molecular weight and volatility in determining release kinetics.

 

Reviewer

When first mentioned, the full Latin name of the plant should be provided – species, author name, and family as well. However, afterwards, the short or common name form should be used throughout the text (T. riparia, line 174…please, check through the text and correct where appropriate).

Answer

The complete form (first mention in the text) is Tetradenia riparia (Hochst.) Codd.

 

 

Reviewer 5 Report

Comments and Suggestions for Authors

In this work, the authors prepared, characterized, and evalutated the antibiofilm effect of free and nanoencapsulated Tetradenia riparia (Hochst) codd leaves essential oil. The content of this study is generally badly written.

The abstract is badly prepared and very confusing. The aim and main content of this work are not clearly introduced. Many sentences are badly structured, such as lines 17-18, lines 25-26. Some abbreviations, such as VERO , are not defined.

Introduction: Conventional antibacterial agents for the treatment of Staphylococcus aureus are not introduced. Why Tetradenia riparia essential oil was selected from various plant sources is not explained.

Line 84-96: What is the purity of the extracted essential oil?

Why PBS was used for the releasing test of essential oil?

Line 139: ”2.9. Strains and Growth Conditions” This content should be combined with other methods.

Some testing such as DSC is not necessary. However, others should be strengthened, such as the release of essential oil in different solvents.

The results are simply discussed which need improvements. The authors should compared their results with previous studies and clearly explain their own results. For example, the physical properties and antibacterial activity of EO and NP are not well discussed.

The position of all figures and tables are inappropriate.

Author Response

REVIEWER 5

All suggestions were considered and addressed in the revised version. Detailed responses are provided below.

Reviewer’s comment:

The content of this study is generally badly written. The abstract is badly prepared and very confusing. The aim and main content of this work are not clearly introduced. Some abbreviations, such as VERO, are not defined.

Response:

The manuscript has undergone comprehensive English language revision, including grammar, sentence structure, and clarity. The abstract was fully rewritten to clearly present the study background, objective, methodology, main results, and conclusions. All abbreviations are now defined at first mention, including VERO (African green monkey kidney epithelial cells).

Reviewer’s comment:

Conventional antibacterial agents for the treatment of Staphylococcus aureus are not introduced. Why Tetradenia riparia essential oil was selected is not explained.

Response:

The Introduction was revised to include a concise overview of conventional antibacterial therapies against Staphylococcus aureus, emphasizing limitations related to resistance and biofilm formation. In addition, the rationale for selecting Tetradenia riparia essential oil was clarified, highlighting its reported antimicrobial and antibiofilm activity and the relevance of its bioactive volatile compounds. Appropriate references were added. Lines 84–96

Reviewer’s comment:

What is the purity of the extracted essential oil? Why PBS was used for the releasing test?

Response:

The essential oil characterization is now clarified. Its composition and relative purity were determined by GC–MS analysis, and the main constituents are reported in the manuscript.PBS was used in the release assay to simulate physiological conditions (pH 7.4), which is relevant for potential biomedical applications. This justification has been explicitly added.

Reviewer’s comment:

Section 2.9 – Strains and Growth Conditions

This content should be combined with other methods.

Response:

This section has been merged with the microbiological assays subsection, improving organization and readability of the Materials and Methods section.

Reviewer’s comment:

Characterization and release studies

Some testing such as DSC is not necessary. Other tests should be strengthened, such as release in different solvents.

Response:

DSC analysis was retained because it provides relevant information on thermal behavior, polymer–oil interactions, and physical stability of the nanoparticles. Its relevance is now better justified in the text.

The discussion of the essential oil release profile was strengthened, with a clearer explanation of the results obtained in PBS and a discussion of limitations regarding other release media.

Reviewer’s comment:

Results and Discussion

The results are simply discussed. Comparisons with previous studies and clearer explanations are needed, particularly for physical properties and antibacterial activity.

Response:

The Results and Discussion section was substantially revised and expanded.

We now include direct comparisons with previous studies regarding nanoparticle size, zeta potential, stability, antibacterial, and antibiofilm activity.

The physical properties and biological performance of free and nanoencapsulated essential oil are now more clealy interpreted and supported by relevant literature.

Reviewer’s comment:

Figures and Tables

The position of all figures and tables are inappropriate.

 Response:

All figures and tables were positioned according to the journal guidelines.

Reviewer 6 Report

Comments and Suggestions for Authors

Dear authors,

Your article reports the preparation of poly(L-lactide) (PLA) nanoparticles containing Tetradenia riparia leaf essential oil (EO) by nanoprecipitation and evaluates antibacterial/antibiofilm activity against Staphylococcus aureus plus cytotoxicity in VERO cells. I would recommend some suggestions, as there are several key experimental/reporting gaps that limit interpretability and translational relevance.

1. You report size by DLS (221.9–396.5 nm) and show SEM/TEM images, but the manuscript should also provide PDI (polydispersity index), number of replicates, and whether intensity/volume/number distributions were used (DLS can look very different depending on the metric).

2. Clarify terminology: you alternately refer to “nanoparticles” and “nanocapsules”; if the system is a matrix nanoparticle vs a core–shell nanocapsule, please justify with evidence.
3. Please extend release duration and fit the data to common kinetic models (e.g., Higuchi, Korsmeyer–Peppas) to support “controlled release” quantitatively.
4. Whether you measured biofilm biomass (e.g., crystal violet) in addition to metabolic activity (MTT). These address different endpoints.

Explicit controls: blank PLA nanoparticles (without EO) in antibiofilm and antibacterial assays, to confirm the polymer/surfactant system does not contribute to activity.

Clarify whether treatments were applied during biofilm formation (prevention) vs to mature biofilms (eradication), since these are not equivalent claims.

5. Table 3 reports MIC/MBC/BIC50 in µg/mL, while you also cite an activity classification in mg/mL Please standardize units and ensure the classification system matches the presented values.

Add statistical analysis (mean ± SD/SEM, n, and significance testing) for MIC/BIC50 where applicable, and for cytotoxicity IC50 (including confidence intervals).

6. If you compute selectivity index (SI = IC50/MIC), please report SI values and interpret them (what SI is considered acceptable for your intended application).
7. The authors mention a stability assay over 30–45 days and cite RDC 45, but the results are not clearly shown (e.g., changes in size/PDI/zeta, EO content, antimicrobial activity over time).

Author Response

REVIEWER 6

Our point-by-point responses are provided below.

Comment 1

You report size by DLS (221.9–396.5 nm) and show SEM/TEM images, but the manuscript should also provide PDI (polydispersity index), number of replicates, and whether intensity/volume/number distributions were used.

Response:

The PDI values reported in this study were obtained directly from the dynamic light scattering (DLS) measurements using the cumulant analysis implemented in the instrument software, in accordance with ISO 22412. The PDI was not manually calculated from the mean particle size and standard deviation.

The reported mean particle size (306.7±7.1 nm) represents the average hydrodynamic diameter, while the PDI reflects the width of the size distribution derived from the autocorrelation function. The obtained PDI values indicated a narrow particle size distribution, confirming the homogeneity of the nanoparticle formulation.

This clarification has been added to the revised manuscript to avoid any ambiguity regarding PDI determination.

Comment 2

Clarify terminology: you alternately refer to “nanoparticles” and “nanocapsules”.

Response:

Based on the nanoprecipitation method employed, the PLA polymeric matrix, and the absence of evidence supporting a distinct core–shell structure, the system is more accurately described as polymeric nanoparticles (matrix-type nanoparticles) rather than nanocapsules

Accordingly, we have standardized the terminology throughout the manuscript to “PLA nanoparticles”, and we now explicitly justify this classification in the Nanoparticle Preparation and Characterization section, supported by SEM/TEM morphological analysis.

Comment 3

Please extend release duration and fit the data to common kinetic models to support “controlled release” quantitatively.

Response:

It is now stated in the text:

1. repiaria essential oil, which contains a higher proportion of sesquiterpenes and diterpenes 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 rapidly (Singh and Pulikkal, 2022).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 interactions (Gupta et al., 2016). Consequently, encapsulated EO 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 effect (Yammine et al., 2024). The controlled release of EO observed in this study is consistent with these established principles, reinforcing the importance of molecular weight and volatility in determining release kinetics.

Comment 4

Whether you measured biofilm biomass (e.g., crystal violet) in addition to metabolic activity (MTT).

Response:

The anti-microbial efficacy of EO and NP against S. aureus biofilms was tested using live/dead staining in combination with SEM analysis and we observed that the essential oil free and nanoencapsulated showed an evident anti-biofilm effect and a prevention of adhesion at sub-MIC concentrations.

Comment 5

Explicit controls: blank PLA nanoparticles (without EO) in antibiofilm and antibacterial assays.

Response:

The results demonstrated that blank nanoparticles did not exhibit significant antibacterial or antibiofilm activity,(data not shown) confirming that the observed effects are attributable to the encapsulated essential oil rather than the polymeric or surfactant system. This clarification has been added to the Materials and Methods and Results sections.

Comment 6

Clarify whether treatments were applied during biofilm formation vs to mature biofilms.

Response:

The antibacterial activity of EO and NP was evaluated against S. aureus ATCC 29213 using broth microdilution assays (Table 3). Samples were classified as follows: MIC ≤ 0.5 mg/mL indicated strong antibacterial activity; 0.6–1.5 mg/mL indicated moderate activity; and MIC > 1.6 mg/mL indicated inactivity [44].

EO and NP exhibited similar antibacterial activity against both planktonic and biofilm forms of S. aureus, indicating that nanoencapsulation did not diminish EO activity. However, enhanced antibacterial activity after encapsulation would have been desirable.

The antibiofilm effects of EO and NP were compared with vancomycin. BIC₅₀ values were 310 µg/mL for EO and 330 µg/mL for NP.

Biofilm morphology after treatment is shown in Figure 4. SEM analysis revealed that untreated cells (A) appeared spherical, dense, and surrounded by abundant extracellular matrix. EO-treated biofilm (B) showed reduced extracellular matrix, altered cell morphology, and fewer cells. NP-treated biofilm (C) also exhibited decreased cell numbers and notable alterations in the cell wall and membrane, suggesting irreversible membrane damage, as previously described by Kang et al. [45].

Comment 7

Table 3 reports MIC/MBC/BIC50 in µg/mL, while you also cite an activity classification in mg/mL Please standardize units and ensure the classification system matches the presented values.

Response

The corrections were made

Comment 8

Add statistical analysis (mean ± SD/SEM, n, and significance testing) for MIC/BIC50 where applicable, and for cytotoxicity IC50 (including confidence intervals).

Response

The data in the tables and figures as described in the footnotes are representative of one out of three independent experiments. For cytotoxicity the IC₅₀ values were obtained by linear regression of dose–response curves generated in R software. Selectivity indices (SI) were calculated as SI = IC₅₀ / MIC.

Comment 9

6. If you compute selectivity index (SI = IC50/MIC), please report SI values and interpret them (what SI is considered acceptable for your intended application).

Response

The selectivity index (SI), defined as the ratio between the IC₅₀ value obtained in VERO cells and the MIC value against Staphylococcus aureus (SI = IC₅₀/MIC), was calculated using the cytotoxicity and antibacterial data presented in Tables 3 and 4.

For free T. riparia essential oil (EO), the IC₅₀ was lower than 125 µg/mL, while the MIC against S. aureus was 125 µg/mL, resulting in an SI value <1. This indicates low selectivity, suggesting that the antibacterial effect of free EO occurs at concentrations close to those inducing cytotoxicity.

In contrast, the nanoencapsulated formulation (NP) showed an IC₅₀ of 533.96 µg/mL and a MIC of 250 µg/mL, corresponding to an SI of approximately 2.1. Although SI values ≥10 are often considered indicative of high therapeutic selectivity for systemic antimicrobial applications, lower SI values may still be acceptable for topical or localized applications, particularly for antibiofilm strategies. Importantly, nanoencapsulation significantly increased the SI compared to free EO, mainly due to the marked reduction in cytotoxicity while maintaining antibacterial and antibiofilm activity.

These results demonstrate that PLA nanoparticle encapsulation improves the therapeutic selectivity of T. riparia EO. The SI values and their interpretation have now been included in the revised manuscript (Results and Discussion sections), and a new sentence was added to clarify the intended application and safety considerations

Comment 10

The authors mention a stability assay over 30–45 days and cite RDC 45, but the results are not clearly shown (e.g., changes in size/PDI/zeta, EO content, antimicrobial activity over time).

Response

It is now stated in the text: Differential scanning calorimetry (DSC) analysis provided valuable insights into the thermal behavior and stability of the polymeric nanoparticles by quantifying the enthalpy changes associated with melting transitions. The DSC parameters are summarized in Table 2. Blank nanoparticles (NPs) exhibited a melting temperature (Tm) of 54.29 °C and a high melting enthalpy (ΔHm) of 429.63 J/g, reflecting a more ordered crystalline structure of the polymeric matrix.

In contrast, nanoparticles loaded with essential oil (EO) showed a slight reduction in melting temperature (52.71 °C) accompanied by a pronounced decrease in melting enthalpy to 115.83 J/g. This substantial reduction in ΔHm indicates a decrease in polymer crystallinity, suggesting that the incorporation of the EO disrupts the crystalline organization of the PLA matrix.

The DSC thermograms presented in Figure 2 corroborate these findings, as blank NPs displayed a larger and more defined endothermic peak, whereas EO-loaded NPs exhibited a markedly reduced peak area. Such thermal behavior indicates strong interactions between the encapsulated EO and the polymeric chains, leading to a more amorphous structure. This structural modification facilitates polymer melting and enhances molecular mobility, which is favorable for controlled and sustained release of the encapsulated EO.

Overall, the DSC results confirm successful encapsulation and demonstrate that EO incorporation modulates the thermal properties of the nanoparticles, supporting their suitability as controlled-release delivery systems for volatile and thermolabile bioactive compounds.

In addition, as described in Section 2.8, a stability assay was conducted over 30–45 days at 25 ± 2 °C, in accordance with RDC 45 (August 9, 2012) [17]. Antibacterial activity was subsequently evaluated using the broth microdilution method following CLSI guidelines. The MIC values of the nanoparticles remained unchanged throughout the 45-day period, indicating that nanoencapsulation preserves EO stability and prevents oxidative degradation.

Reviewer 7 Report

Comments and Suggestions for Authors

The work titled " Preparation, Characterization, and Antibiofilm Effect of Free and Nanoencapsulated Tetradenia riparia (Hochst) Codd Leaves Essential Oil" represents novel work of nanoencapsulation which is an alternative way that improves stability, reduces toxicity and controls the release of oil. The results showed that EO and NP show antibacterial and antibiofilm activity against S. aureus. NP was less cytotoxic than EO. Nanoparticle prevented rapid EO evaporation and degradation and enhanced its stability. NP stability was studied using zeta potential. Its value was determined to be around -23.1 mV, which indicates that NP are in fact stable. Melting temperature and melting enthalpy for Blank NP were 54.29 °C and 429.63 J/g. The decreasing in melting enthalpy from 429.63 to 115.83 J/g in NP containing EO makes this system favorable to controlled release of essential oils. NP has a smaller area under the peak, indicating that the EO may modify the crystalline organization, facilitating melting and thus the release of EO. EO and NP presented a growth inhibition of planktonic and biofilm formation against S. aureus. NP were less cytotoxic than free EO. Thus, these findings may contribute to the development of new strategies against infections caused by S. aureus. All of these findings of this work seem valuable and suitable contribution to be published in future Pharmacology Journal after justifying the following major points:

• It is recommended to reduce the similarity and overlapping rate which seems around 34%
• It is recommended to change the starting sentence in the abstract to be aligned more with journal scope  
• It is recommended too to add the used methods in this work to the abstract
• Line 26 what did you mean with EO.aure??
• The main results values like IC50 or MIC could be added to the abtract to be more attractive
• Improve the conclusion of this abstract work more
• The aim of this work should be improved in the lines 71-73
• The plants’ leaves were collected before 10 years, can you explain this point in the discussion section (2015)
• All used chemicals and reagents should be written in the method section
• The authors should add references to each method section since the methods are not novel
• A statistical analysis section should be added to the method section accordingly
• The tables and figures should be moved to the site after the paragraph mentioned them directly
• The title of Table 1 should be corrected to GC-MS not CG-MS
• The IC50 should be edited as 50 subscripts in the whole manuscript

The conclusion section should be improved

Best wishes

Author Response

REVIEWER 7

Response to Reviewer

All comments were carefully addressed as detailed below.

Reviewer comment:

It is recommended to reduce the similarity and overlapping rate which seems around 34%.

Response:

The manuscript has been carefully revised to reduce similarity and textual overlap. Several sections were rewritten, particularly the Introduction, Methods, and Discussion, to ensure originality and compliance with journal standards. As a result, the overall similarity index was substantially reduced.

Reviewer comment:

It is recommended to change the starting sentence in the abstract to be aligned more with journal scope.

Response:

The opening sentence of the abstract was revised to better align with the journal’s pharmacological and biomedical scope, emphasizing the relevance of nanoencapsulation strategies in antimicrobial and antibiofilm applications.

Reviewer comment:

It is recommended too to add the used methods in this work to the abstract.

Response:

The abstract was updated to briefly include the main methodologies employed, such as nanoprecipitation for nanoparticle preparation, physicochemical characterization (DLS, zeta potential, DSC), antibacterial and antibiofilm assays, and cytotoxicity evaluation in VERO cells.

Reviewer comment:

Line 26 what did you mean with EO.aure??

Response:

The term “EO.aure” was a typographical error and has been deleted

Reviewer comment:

The main results values like IC₅₀ or MIC could be added to the abstract to be more attractive.

Response:Key quantitative results, including MIC and IC₅₀ values for free EO and nanoencapsulated EO, were added to the abstract to enhance its scientific impact and clarity.

Reviewer comment:

Improve the conclusion of this abstract work more.

Response:

The concluding sentence of the abstract was rewritten to better highlight the significance of the findings, emphasizing the potential of PLA nanoparticles as a promising strategy to improve the stability, safety, and antibiofilm efficacy of essential oils against S. aureus.

Reviewer comment:

The aim of this work should be improved in the lines 71–73.

Response:

The aim of the study was rewritten to be clearer and more objective, explicitly stating the preparation, physicochemical characterization, antibacterial/antibiofilm evaluation, and cytotoxicity assessment of free and nanoencapsulated Tetradenia riparia essential oil.

Reviewer comment:

The plants’ leaves were collected before 10 years, can you explain this point in the discussion section (2015).

Response:

An explanation was added to the Discussion section clarifying that the essential oil was properly stored under controlled conditions (protected from light, oxygen, and high temperatures), which ensured its chemical stability and preserved its bioactivity over time, as supported by literature.

Reviewer comment:

All used chemicals and reagents should be written in the method section.

Response:

The Materials and Methods section was revised to include a complete list of all chemicals and reagents used, along with their suppliers and purity grades.

Reviewer comment:

The authors should add references to each method section since the methods are not novel.

Response:

Appropriate references were added to all experimental procedures, including nanoparticle preparation, physicochemical characterization, antimicrobial assays, antibiofilm evaluation, cytotoxicity testing, and thermal analysis.

Reviewer comment:

A statistical analysis section should be added to the method section accordingly.

Response:

A dedicated Statistical Analysis subsection was added to the Methods section, describing the statistical tests used, significance levels, and data presentation format.

Reviewer comment:

 The tables and figures should be moved to the site after the paragraph mentioned them directly.

Response:

All tables and figures were organized and positioned in accordance with the journal’s formatting guidelines.

Reviewer comment:

The title of Table 1 should be corrected to GC-MS not CG-MS.

Response:

This error has been corrected. The title of Table 1 now correctly reads GC–MS.

Reviewer comment:

The IC50 should be edited as 50 subscripts in the whole manuscript.

Response:
All occurrences of IC50 were corrected to IC₅₀ throughout the manuscript.

Reviewer comment:

The conclusion section should be improved.

Response:

The Conclusion section was expanded and strengthened to clearly summarize the main findings, highlight the advantages of nanoencapsulation over free essential oil, and emphasize the potential pharmacological and antimicrobial applications of the developed nanoparticles.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

With the questions and suggestions having been answered, I therefore propose the publication of this study.

Reviewer 5 Report

Comments and Suggestions for Authors

The quality of the manuscript has been improved a lot. The authors should pay attention to the position of all figures and tables. Meanwhile, the amount of wording duplication in the original manuscript is too high (34%). The editor needs to check the wording duplication of the revised manuscript before publication.

Reviewer 6 Report

Comments and Suggestions for Authors

The revised version is Ok

Reviewer 7 Report

Comments and Suggestions for Authors

i hope to receive a clean version of the last version of the manuscript