Membrane-Mediated Nanoassembly of Lysozyme–Tannic Acid for Crystallization-Suppressed Nobiletin Delivery: Enhanced Cellular Uptake and Mucus Penetration

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors presented an interesting manuscript entitled Membrane-Mediated Nanoassembly of Lysozyme-Tannic Acid for Crystallization-Suppressed Nobiletin Delivery: Enhanced Cellular Uptake and Mucus Penetration, which require further improvement following given suggestions.

1. The manuscript clearly demonstrates the optimization of lysozyme-tannic acid nanoparticles for nobiletin delivery, however the introduction could be strengthened by explicitly comparing the proposed system with other polyphenol-protein nano-assembly approaches, highlighting unique advantages or mechanisms.
2. The rationale for selecting lysozyme and tannic acid is well justified, but consider adding a brief discussion on the potential immunogenicity or in vivo safety of lysozyme, especially in the context of oral delivery, to address translational concerns.
3. Though the methodology for nanoparticle characterization is comprehensive, suggested to include a table summarizing the key physicochemical parameters under optimal and suboptimal conditions to facilitate rapid comparison by readers.
4. Although the mechanistic studies using QCM-D, ITC, and VT-FTIR are robust, though the manuscript would benefit from a schematic illustration integrating these findings to clarify the multivalent weak-interaction network driving nanoparticle formation and stability.
5. The transmembrane permeation experiments are well designed, however the discussion could be expanded to address how the energy-independent passive diffusion mechanism of LT-NOB may overcome common intestinal absorption barriers, such as P-gp efflux, compared to conventional delivery systems.
6. The Caco-2HT29-MTX co-culture model provides valuable insight into mucus penetration, possibly including a direct comparison of mucus layer penetration depth and nanoparticle retention time with other reported nano-carriers to contextualize the findings.​
7. The manuscript elaborated future research directions, however adding a brief section on potential scalability and manufacturing challenges for lysozyme-tannic acid nanoparticles, which would help bridge the gap between laboratory-scale findings and clinical translation.
8. The conclusion effectively summarizes the key findings, however consider emphasizing the broader implications of this work for the delivery of other hydrophobic drugs or bioactive compounds, beyond nobiletin, to highlight its generalizability.

 

Author Response

Dear Reviewers,

We sincerely appreciate the constructive comments from the reviewers of the Journal of Biomolecules. These suggestions have significantly enhanced the quality of our manuscript. We have carefully addressed each point raised and provide detailed responses below.（Please note that the revisions to the figures are not reflected in these comments. The complete version of our response is provided in the attached file, which we kindly invite the reviewer to refer to.）

Reviewer 1:

The authors presented an interesting manuscript entitled Membrane-Mediated Nanoassembly of Lysozyme-Tannic Acid for Crystallization-Suppressed Nobiletin Delivery: Enhanced Cellular Uptake and Mucus Penetration, which require further improvement following given suggestions.

Comment 1:The manuscript clearly demonstrates the optimization of lysozyme-tannic acid nanoparticles for nobiletin delivery, however the introduction could be strengthened by explicitly comparing the proposed system with other polyphenol-protein nano-assembly approaches, highlighting unique advantages or mechanisms.

Response:

Based on your suggestions, I have revised the Introduction section as follows:...structured nanocarriers for the delivery of functional factors^[6]. However, many existing polyphenol-protein nano-assemblies often rely on a dominant single interaction or require multi-step processing. For example, lactoferrin-EGCG complexes primarily utilize electrostatic and hydrophobic interactions, which may demand precise control over environmental conditions for optimal assembly. Similarly, while TA can effectively crosslink with various proteins like BSA, such systems frequently emphasize the cargo-delivery function of the protein itself, rather than leveraging a synergistic, multi-mechanistic assembly network that concurrently inhibits drug crystallization. In contrast, the lysozyme-tannic acid (Lys-TA) system proposed herein capitalizes on a cascade of well-orchestrated interactions: the strong electrostatic attraction initiates rapid complexation, which is subsequently stabilized by a network of hydrogen bonding and hydrophobic effects. This multi-valent weak interaction paradigm, as detailed in our mechanistic studies (Sections 3.3), is crucial for suppressing the crystallization of encapsulated nobiletin. Furthermore, the entire nanoparticle formation is achieved through a straightforward one-step co-assembly process under mild conditions, avoiding the need for chemical crosslinkers or complex purification steps commonly associated with engineered protein carriers, compared to the limitations of traditional protein carriers^[7]. Compared to the limitations of traditional protein carriers (e.g., whey protein, human serum albumin, or transferrin), such as complex preparation processes (requiring chemical crosslinking or genetic engineering modification) and high production costs (cumbersome recombinant protein purification procedures), lysozyme exhibits significant advantages:..

Location: lines 76-93

 

Comment 2: The rationale for selecting lysozyme and tannic acid is well justified, but consider adding a brief discussion on the potential immunogenicity or in vivo safety of lysozyme, especially in the context of oral delivery, to address translational concerns.

Response:

Based on your suggestions, I have revised the Introduction section as follows:

...(3) Functional integration: Inherent lytic activity hydrolyzes peptidoglycan in Gram-positive bacteria, conferring antimicrobial functionality to the carrier^[8]. (4) Favorable Safety Profile for Delivery: Lysozyme is an endogenous natural protein that is classified as Generally Recognized As Safe (GRAS) for food applications. It possesses inherently low immunogenicity, which establishes a crucial foundation for its biosafety as a delivery vehicle^[9]. ...

Location: lines 103-106

 

Comment 3: Though the methodology for nanoparticle characterization is comprehensive, suggested to include a table summarizing the key physicochemical parameters under optimal and suboptimal conditions to facilitate rapid comparison by readers.

Response:

In accordance with your suggestions, I have revised the relevant section accordingly. The modifications are presented below:

2.2.2 Measurement of Encapsulation Efficiency and Drug Loading

To provide a comprehensive overview of the system's performance, the key physicochemical characteristics (including particle size, PDI, and zeta potential) and drug-encapsulation metrics (EE% and DL%) under the optimized parameters are systematically compared with those observed under suboptimal conditions in Table 1. This quantitative summary highlights the critical impact of parameter deviations on nanoparticle integrity and reinforces the superiority of the established synthesis protocol (Lys 8 mg/mL, pH 7.4, NOB 5 mg/mL) in achieving high monodispersity and maximal drug loading.

Table 2. Comparison of physicochemical properties of LT-NOB nanoparticles under optimal and suboptimal synthesis conditions.

Parameters	Optimal Conditions	Suboptimal Conditions (Deviations)	Impact of Deviation
Synthesis	Lys: 8 mg/mLpH: 7.4NOB: 5 mg/mL	Lys: < 8 mg/mL or > 8 mg/mLpH: Acidic (<7.0) or Alkaline (>8.0)NOB: Deviated from 5 mg/mL	Lys Deviation: Reduced encapsulation efficiency (EE%) and drug loading (DL%) .pH Deviation: Weakened electrostatic or hydrophobic interactions.
Variables Particle Size (Diameter)	212 nm (Uniform distribution)	Significantly Increased or Broadened distribution	pH deviation leads to aggregation or large precipitates due to reduced binding affinity.
Polydispersity Index (PDI)	0.03 (High monodispersity)	> 0.3	Indicates poor dispersion and system instability.
Zeta Potential	-17 mV	Varies (e.g., reduced negative charge magnitude)	Less stable colloidal system prone to aggregation.
Encapsulation Efficiency (EE%)	89.50%	< 89.5%	Lower Lys concentration results in insufficient wall material; extreme pH disrupts assembly forces.
Drug Loading (DL%)	47.25%	Lower	Excessive Lys (12 mg/mL) increases shell thickness without adding drug payload.
Morphology & Crystallinity	Regular spherical shape; Amorphous NOB (Crystallization suppressed)	Irregular shape; Crystalline precipitates observed	Appearance of birefringence in POM indicates failure to inhibit NOB crystallization.

Location: lines 500-501

 

Comment 4: Although the mechanistic studies using QCM-D, ITC, and VT-FTIR are robust, though the manuscript would benefit from a schematic illustration integrating these findings to clarify the multivalent weak-interaction network driving nanoparticle formation and stability.

Response:

In accordance with your suggestions, I have revised the relevant section accordingly. The modifications are presented below:

3.3.3 Temperature-Dependent Infrared Spectroscopy Reveals Molecular Conformational Changes

Collectively, these spectroscopic and thermodynamic analyses elucidate the multi-scale assembly mechanism of the LT-NOB system. As systematically illustrated in Fig. 6, the assembly is initiated by the formation of a viscoelastic and compact composite film under neutral pH (QCM-D), stabilized by a synergistic network of electrostatic, hydrogen bonding, and hydrophobic interactions (ITC), and finally locked into a thermally stable β-turn-dominated conformation that effectively suppresses drug crystallization (VT-FTIR).

 

Fig.6. Schematic illustration of the assembly mechanism and structure of LT-NOB nanoparticles at pH 7.4

Location: lines 623-631

 

Comment 5: The transmembrane permeation experiments are well designed, however the discussion could be expanded to address how the energy-independent passive diffusion mechanism of LT-NOB may overcome common intestinal absorption barriers, such as P-gp efflux, compared to conventional delivery systems.

Response:

In accordance with your suggestions, I have revised the relevant section accordingly. The modifications are presented below:

3.4.2.2 Mechanistic Investigation of Transport Pathways

... In summary, fundamental differences exist between the transmembrane transport mechanisms of LT-NOB and free NOB: LT-NOB achieves efficient uptake through energy-independent passive diffusion (ER = 1.3), whereas free NOB transport significantly relies on ATP-driven active transport processes (baseline ER = 0.78, ER = 0.39 post-Verapamil treatment)^[32]. In summary, fundamental differences exist between the transmembrane transport mechanisms of LT-NOB and free NOB: LT-NOB achieves efficient uptake through energy-independent passive diffusion (ER = 1.3), whereas free NOB transport significantly relies on ATP-driven active transport processes. Crucially, this passive diffusion mechanism confers a significant advantage in overcoming multidrug resistance barriers. Unlike free NOB, which serves as a substrate for P-gp and is actively pumped out of the enterocytes, the LT-NOB nanocomplex effectively 'masks' the drug within its core/shell structure. By preventing direct contact between the drug molecules and the P-gp efflux binding sites on the apical membrane, the nanoparticle circumvents the active efflux trap. Consequently, the absorption of LT-NOB is driven solely by the concentration gradient rather than being limited by the saturation kinetics or abundance of efflux transporters, a limitation frequently encountered in conventional drug delivery systems. ...

Location: lines 784-797

 

Comment 6: The Caco-2HT29-MTX co-culture model provides valuable insight into mucus penetration, possibly including a direct comparison of mucus layer penetration depth and nanoparticle retention time with other reported nano-carriers to contextualize the findings.​

Response:

In accordance with your suggestions, I have revised the relevant section accordingly. The modifications are presented below:

3.4.3 Comparison of Permeation Behavior Between Caco-2/HT29-MTX Co-Culture and Caco-2 Monolayer Models

To more accurately simulate human intestinal physiology, this study further employed a Caco-2/HT29-MTX co-culture model (containing 90% Caco-2 intestinal epithelial cells and 10% mucus-secreting HT29-MTX cells) to investigate the transmembrane transport properties of LT-NOB. As shown in Fig. 9D, the 2 h cumulative permeation of LT-NOB in the co-culture system reached 1.21 μg, exceeding the 0.89 μg observed in the Caco-2 monolayer model. CLSM 3D reconstruction results (Fig. 9E) revealed significantly enhanced axial penetration depths (XZ/YZ planes) of LT-NOB in the Caco-2/HT29-MTX co-culture layer compared to the Caco-2 monolayer model. This superior penetrability can be attributed to the unique surface properties of the LT-NOB assembly. Unlike conventional cationic nanocarriers (e.g., chitosan-based systems) that are often immobilized in the superficial mucus layer due to strong electrostatic attraction with negatively charged mucin fibers , the LT-NOB nanoparticles exhibit a moderate negative zeta potential (-17 mV)  and a hydrophilic polyphenol coating. These characteristics confer "mucus-inert" properties, effectively minimizing steric obstruction and adhesive entrapment. Consequently, LT-NOB achieves a deeper axial penetration depth and avoids the "mucoadhesive trap" common to traditional carriers, thereby facilitating closer contact with the underlying epithelial cells for enhanced absorption^[35].

Location: lines 808-818

 

Comment 7: The manuscript elaborated future research directions, however adding a brief section on potential scalability and manufacturing challenges for lysozyme-tannic acid nanoparticles, which would help bridge the gap between laboratory-scale findings and clinical translation.

Response:

In accordance with your suggestions, I have revised the conclusion. The modifications are presented below:

...system in in vivo models.Crucially, the inherent design of the LT-NOB system offers a strategic advantage for bridging laboratory findings with industrial and clinical applications. Its reliance on a 'green', one-step aqueous assembly process aligns seamlessly with the growing industrial demand for sustainable and cost-effective manufacturing. Moreover, the exclusive use of biocompatible, naturally derived components (GRAS-grade materials) significantly lowers safety concerns and regulatory hurdles associated with synthetic carriers. These attributes establish LT-NOB not merely as a delivery vehicle, but as a versatile platform with high translational potential for developing next-generation functional foods and pharmaceutical formulations.

Location: lines 865-873

 

Comment 8: The conclusion effectively summarizes the key findings, however consider emphasizing the broader implications of this work for the delivery of other hydrophobic drugs or bioactive compounds, beyond nobiletin, to highlight its generalizability.

Response:

In accordance with your suggestions, I have revised the conclusion. The modifications are presented below:

...delivery of hydrophobic drugs. The core findings—regarding the stabilization of amorphous drugs via multivalent weak interactions, the promotion of cellular uptake through macropinocytosis, the facilitation of lysosomal escape and ER targeting, and the enhancement of mucus penetration and P-gp evasion—collectively outline a versatile and generalizable platform technology. This Lys-TA nanoassembly platform holds considerable promise for adapting to and improving the bioavailability of numerous other poorly water-soluble drugs and nutraceuticals beyond nobiletin. ...

Location: lines 850-856

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript contributes to the important field of enhancement of hydrophobic drugs bioavailability. The authors reported results of numerous methods to prove their concept of using lysozyme-tannic acid complex as a carrier for nobiletin (NOB). Although experimental part is described in details, some important issues remained unclear:

 1.Mass ratio between components Lysozyme -tannic acid-NOB mentioned in different parts of the manuscript is ambiguous.

 From Lines 89-93 it can be concluded that lysozyme solution (40 μL) with concentrations (2, 4, 6, 8, 10, 12 mg/mL) + stock solutions of TA (20 μL, 40 mg/mL) were added to the 10 ml of buffer containing NOB. Thus, lysozyme will be diluted by the factor 250. At maximal lysozyme concentration the ratio in final solution will be: lysozyme 0.048 mg/l, tannic acid  0.08 mg/l,  NOB 0.5 mg/L. So the content of LT is too low for NOB encapsulation. Further, the author mention lysozyme concentrations up to 12 mg/mL, as if this is not stock solution but resulting concentration after mixing with buffer: line Line 347 says (Lys) concentration (2-12 mg/mL), pH (5.0, 6.0, 7.0, 7.4, 8.0), and nobiletin (NOB) concentration (4-7 mg/mL). Please make it clear, maybe in the form of table with notations for all systems and mass ratio of the components. This is important also to understand drug loading capacity: Lys-TA complex stock solution 1 mg/mL (line 206). Description of systems compositions in Supplementary materials 1.1 и 1.2 is also ambiguous, TEM images of NOB without LT is required for the reference.

2. It is not clear, what is drug loading, %. What is the difference between drug loading and encapsulation efficiency? Please add formula for calculations to the section 2.4. Measurement of Encapsulation Efficiency and Loading Capacity.
3. It is not clear, why Coumarin-6 (green fluorescent tracer) was added together with NOB for confocal microscopy and flow cytometry. FITC channel is shown in Fig. 6. While NOB autofluorescence (Ex/Em = 405/450 nm) is not FITC. Which fluorescence intensity is shown in this figure? Please add example of gating in flow cytometry analysis. Scale bar is missing in Fig.6C
4. Scale bar is missing in Fig. 8E. Is it fluorescence in FITC channel?
5. What means film-mediated nanoassembly (line 684), if theNOB encapsulation occurred in solution?
6. It is not clear what was the physicochemical background for optimal encapsulation conditions? How zeta potential of LT changes with pH and ratio of the components? How solubility of NOB changes with pH?

Author Response

Dear Reviewers,

We sincerely appreciate the constructive comments from the reviewers of the Journal of Biomolecules. These suggestions have significantly enhanced the quality of our manuscript. We have carefully addressed each point raised and provide detailed responses below.（Please note that the revisions to the figures are not reflected in these comments. The complete version of our response is provided in the attached file, which we kindly invite the reviewer to refer to.）

Reviewer 2:

The manuscript contributes to the important field of enhancement of hydrophobic drugs bioavailability. The authors reported results of numerous methods to prove their concept of using lysozyme-tannic acid complex as a carrier for nobiletin (NOB). Although experimental part is described in details, some important issues remained unclear:

Comment 1: Mass ratio between components Lysozyme -tannic acid-NOB mentioned in different parts of the manuscript is ambiguous.From Lines 89-93 it can be concluded that lysozyme solution (40 μL) with concentrations (2, 4, 6, 8, 10, 12 mg/mL) + stock solutions of TA (20 μL, 40 mg/mL) were added to the 10 ml of buffer containing NOB. Thus, lysozyme will be diluted by the factor 250. At maximal lysozyme concentration the ratio in final solution will be: lysozyme 0.048 mg/l, tannic acid  0.08 mg/l,  NOB 0.5 mg/L. So the content of LT is too low for NOB encapsulation. Further, the author mention lysozyme concentrations up to 12 mg/mL, as if this is not stock solution but resulting concentration after mixing with buffer: line Line 347 says (Lys) concentration (2-12 mg/mL), pH (5.0, 6.0, 7.0, 7.4, 8.0), and nobiletin (NOB) concentration (4-7 mg/mL). Please make it clear, maybe in the form of table with notations for all systems and mass ratio of the components. This is important also to understand drug loading capacity: Lys-TA complex stock solution 1 mg/mL (line 206). Description of systems compositions in Supplementary materials 1.1 и 1.2 is also ambiguous, TEM images of NOB without LT is required for the reference.

Response:

In accordance with your suggestions, I have revised the relevant section accordingly. The modifications are presented below:

①A new table has been added.

Table 1. Detailed composition and mass ratios of the LT-NOB nanocomplex preparation under optimal and experimental conditions.

 

Component	Role	Stock Concentration (Cstock)	Volume Added (Vadded)	Total Mass Input (minput)	Remarks (Experimental Range)
Nobiletin (NOB)	Functional Cargo (Core)	5 mg/mL	1.0 mL	5.00 mg	Fixed at 5 mg/mL during Lys optimization; varied (4–7 mg/mL) in drug conc. studies.
Tannic Acid (TA)	Crosslinker / Shell	40 mg/mL	20 μL	0.80 mg	Fixed concentration and volume throughout the study.
Lysozyme (Lys)	Wall Material / Shell	8 mg/mL (Optimal)	40 μL	0.32 mg	Stock concentration varied from 2 to 12 mg/mL (corresponding to 0.08–0.48 mg mass input).
Buffer (MOPS)	Solvent Medium	0.01 M (pH 7.4)	9.0 mL	-	pH varied from 5.0 to 8.0 during optimization.

Location: lines 148-149

②To enhance clarity, the preparation method in this section has been supplemented with the following details.

2.2.1 Preparation of the LT-NOB

To investigate the effect of Lys concentration on the self-assembly behavior of the ternary complex nanoparticles, a series of complexes were constructed by varying the Lys concentration. First, gradient solutions of Lys (2, 4, 6, 8, 10, 12 mg/mL) were prepared, alongside stock solutions of TA (40 mg/mL) and NOB (5 mg/mL). Under continuous magnetic stirring (600 rpm), 1 mL of the NOB stock solution was transferred to a sample vial containing 9 mL of 0.01 M MOPS buffer (pH 7.4). Subsequently, 20 μL of TA solution and 40 μL of Lys solution were rapidly injected sequentially into this system. This resulted in final concentrations in the 10 mL reaction system as follows: NOB at 0.5 mg/mL, TA at 0.08 mg/mL, and Lys ranging from 0.008 to 0.048 mg/mL (corresponding to the 2-12 mg/mL stock solutions). Stirring was continued for several seconds after injection, ultimately yielding LT-NOB ternary nanocomplex systems with increasing Lys concentration gradients. Preparation processes under different environmental conditions are in Supplementary Methods 1.

Location: lines 148-151

③A TEM image of NOB has been added to the Supplementary Materials.

 

Location: Supplementary Information (Figure S6)

 

Comment 2: It is not clear, what is drug loading, %. What is the difference between drug loading and encapsulation efficiency? Please add formula for calculations to the section 2.4. Measurement of Encapsulation Efficiency and Loading Capacity.

Response:

In accordance with your suggestions, I have revised the relevant section accordingly. The modifications are presented below:

The Encapsulation Efficiency (EE%) and Drug Loading (DL%) were calculated based on the quantitative analysis of free NOB in the supernatant. EE% represents the percentage of total NOB that was successfully entrapped within the nanoparticles, while DL% indicates the mass percentage of encapsulated NOB relative to the total mass of the nanoparticles. The specific calculation formulas are as follows:

             EE(%)=[(Wtotal-Wfree)/Wtotal]x100%                 (1)

DL(%)=[(Wtotal-Wfree)/Wtotal_NP]x100%            (2)

(Where Wtotal is the total mass of NOB added initially, Wfree is the mass of free NOB detected in the supernatant, and Wtotal_NP is the total weight of the freeze-dried LT-NOB nanoparticles.)

Location: lines 222-231

 

Comment 3: It is not clear, why Coumarin-6 (green fluorescent tracer) was added together with NOB for confocal microscopy and flow cytometry. FITC channel is shown in Fig. 6. While NOB autofluorescence (Ex/Em = 405/450 nm) is not FITC. Which fluorescence intensity is shown in this figure? Please add example of gating in flow cytometry analysis. Scale bar is missing in Fig.6C.

Response:

Rationale for using Coumarin-6 (C6): Although Nobiletin (NOB) exhibits autofluorescence (Ex/Em = 405/450 nm), its fluorescence intensity is relatively weak and prone to photobleaching, which challenges the acquisition of high-quality images for intracellular tracking. To ensure robust visualization and accurate quantification of cellular uptake, we encapsulated Coumarin-6 (C6) into the nanoparticles. C6 is a standard hydrophobic fluorescent probe widely used to mimic lipophilic drugs (like NOB). It shares similar physicochemical properties with NOB but offers significantly higher quantum yield and photostability (detected in the FITC channel).

Clarification of Figure 6: Consequently, the fluorescence intensity shown in Figure 6 corresponds to the C6-loaded nanoparticles (green), not NOB itself.

Missing Information: We have added the scale bar to Figure 6C and included the representative gating strategy for flow cytometry in the Supplementary Information (Figure S7) as requested.

 

Comment 4: Scale bar is missing in Fig. 8E. Is it fluorescence in FITC channel?

Response:

Fig. 8. ... (E) Penetration layer of LT-NOB across the Caco-2 cell monolayer and the Caco-2/HT29-MTX co-cultured cell layer observed by CLSM. Green fluorescence indicates Coumarin-6 labeled LT-NOB nanoparticles collected in the FITC channel. Scale bar = 10 μm.

 

Location: line 824

 

Comment 5: What means film-mediated nanoassembly (line 684), if theNOB encapsulation occurred in solution?

Response:

This study establishes a novel interfacial film-mediated nanoassembly strategy...  The term 'film-mediated' refers to the formation of a dense, viscoelastic Lys-TA composite shell around the drug core. Although the assembly occurs in bulk solution, the mechanism is driven by the continuous deposition of Lys-TA complexes at the solid-liquid interface of the hydrophobic drug nuclei, forming a protective 'film' or membrane analogous to the macroscopic films characterized by QCM-D.

 

Comment 6: It is not clear what was the physicochemical background for optimal encapsulation conditions? How zeta potential of LT changes with pH and ratio of the components? How solubility of NOB changes with pH?

In response to your question, I provide the following explanation:

Response:

1. Zeta Potential vs. pH and Ratio:

The optimization is governed by the electrostatic matching between Lys (pI ≈ 11.0) and TA (pKa ≈ 8.5).

Effect of pH: As shown in Fig. 1B and the QCM-D data (Fig. 3), at pH 7.4, TA is partially deprotonated, providing sufficient negative charges to bind with positively charged Lys. This results in a stable nanocomplex with a Zeta potential of -17 mV. Lower pH (<6) leads to protonation of TA and weak binding, while higher pH (>8) reduces the positive charge of Lys.

Effect of Ratio: Increasing the Lys concentration shifts the Zeta potential towards positive values due to charge neutralization. The optimal ratio (Lys 8 mg/mL) achieves a charge balance that prevents both aggregation (caused by near-zero charge) and loose assembly.

2. NOB Solubility vs. pH:

We clarified that Nobiletin (NOB) is a polymethoxylated flavone lacking ionizable phenolic hydroxyl groups. Therefore, its intrinsic solubility is pH-independent within the investigated range (pH 5–8). The superior encapsulation at pH 7.4 is carrier-driven, not drug-driven. It stems from the formation of the most compact and viscoelastic Lys-TA shell (as confirmed by QCM-D and VT-FTIR) at this pH, which effectively entraps the hydrophobic NOB core and suppresses its crystallization.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

this manuscript on the self-assembly of tannic acid/nobiletin in the presence of lysozyme and viability of these constructs as therapeutic carriers looks to be a valuable contribution to the field of drug delivery and encapsulation. the authors have done a thorough job exploring the fundamental chemical and physical interactions that dictate self-assembly behaviors and in this study have provided a framework to understand and evaluate the performance of similar systems. they have also carried the momentum forward to address cytotoxicity and characterize internalization pathways for these carriers. this manuscript represents solid work carried out with the proper level of depth and rigor and the scientific approach gives confidence to the readers. there are some minor issues with data presentation that should be straightforward for the authors to address:

plots in figure 1b should have the axis title "zeta potential" instead of "zata potential"

figure 1 is too dense and the scale bars and confocal images are too small to be of practical use to readers.

figure 2b needs appropriate x axis labels to indicate what the concentrations represent

the authors should explain the difference between loading efficiency and encapsulation efficiency since the method for calculating these values is not given in the experimental section

figure 3 has the same crowding issue where the labels above the bar plot are too small to be of practical value

the authors should evaluate the density of all figures since they all tend toward being too crowded and making labels difficult to interpret

 

Author Response

Dear Reviewers,

We sincerely appreciate the constructive comments from the reviewers of the Journal of Biomolecules. These suggestions have significantly enhanced the quality of our manuscript. We have carefully addressed each point raised and provide detailed responses below.（Please note that the revisions to the figures are not reflected in these comments. The complete version of our response is provided in the attached file, which we kindly invite the reviewer to refer to.）

Reviewer 3:

this manuscript on the self-assembly of tannic acid/nobiletin in the presence of lysozyme and viability of these constructs as therapeutic carriers looks to be a valuable contribution to the field of drug delivery and encapsulation. the authors have done a thorough job exploring the fundamental chemical and physical interactions that dictate self-assembly behaviors and in this study have provided a framework to understand and evaluate the performance of similar systems. they have also carried the momentum forward to address cytotoxicity and characterize internalization pathways for these carriers. this manuscript represents solid work carried out with the proper level of depth and rigor and the scientific approach gives confidence to the readers. there are some minor issues with data presentation that should be straightforward for the authors to address:

Comment 1: plots in figure 1b should have the axis title "zeta potential" instead of "zata potential"

Response:

The spelling error in Figure 1 has been corrected.

 

 

Comment 2: figure 1 is too dense and the scale bars and confocal images are too small to be of practical use to readers.

Response:

As suggested, Figure 1 has been revised accordingly (see below). Furthermore, the particle size distribution plot and the CLSM (confocal laser scanning microscopy) image have been moved to the Supplementary Materials.

 

 

Comment 3: figure 2b needs appropriate x axis labels to indicate what the concentrations represent

Response:

As requested, the X-axis label has been added to Figure 2b.

 

 

Comment 4: the authors should explain the difference between loading efficiency and encapsulation efficiency since the method for calculating these values is not given in the experimental section.

Response:

In accordance with your suggestions, I have revised the relevant section accordingly. The modifications are presented below:

The Encapsulation Efficiency (EE%) and Drug Loading (DL%) were calculated based on the quantitative analysis of free NOB in the supernatant. EE% represents the percentage of total NOB that was successfully entrapped within the nanoparticles, while DL% indicates the mass percentage of encapsulated NOB relative to the total mass of the nanoparticles. The specific calculation formulas are as follows:

             EE(%)=[(Wtotal-Wfree)/Wtotal]x100%                 (1)

DL(%)=[(Wtotal-Wfree)/Wtotal_NP]x100%            (2)

(Where Wtotal is the total mass of NOB added initially, Wfree is the mass of free NOB detected in the supernatant, and Wtotal_NP is the total weight of the freeze-dried LT-NOB nanoparticles.)

Location: lines 227-236

 

Comment 5: figure 3 has the same crowding issue where the labels above the bar plot are too small to be of practical value

Response:

In accordance with your suggestions, I have revised the Fig.3. The modifications are presented below:

 

 

Comment 6: the authors should evaluate the density of all figures since they all tend toward being too crowded and making labels difficult to interpret

Response:

As recommended, the overly crowded layout of Figures 1 and 3 has been corrected.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have reflected all the said suggestions and comments, which made the manuscript enhanced with improved readability; Thus, I suggest for further consideration with acceptance.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript was improved and  now can be accepted for publication