Nano-Encapsulated Spicule System Enhances Delivery of Wharton’s Jelly MSC Secretome and Promotes Skin Rejuvenation: Preclinical and Clinical Evaluation

1. Summary

Thank you very much for taking the time to review this manuscript.

The detailed responses, along with the corresponding revisions and corrections, are highlighted below.

On behalf of all authors, we would like to sincerely thank you and the reviewers for the constructive comments and valuable suggestions on our manuscript entitled “Nano-Encapsulated Spicule Delivery Enhances Delivery of Wharton’s Jelly MSC Secretome and Skin Rejuvenation in Preclinical and Clinical Evaluation” (Manuscript ID: ijms-3914486), submitted to the International Journal of Molecular Sciences (Special Issue: Roles and Function of Extracellular Vesicles in Diseases: 3rd Edition).

We have carefully revised the manuscript according to the reviewers’ and editorial office’s instructions.

A detailed, point-by-point response to the reviewers’ comments is provided in the attached response file.

Comments 1: Institutional Review Board (IRB) Approval Dates

•  Response 1: [p.17, lines 516-518]
• We have added the approval dates for the IRB numbers as requested
• Revised text (Institutional Review Board Statement):

“This study involving human tissue and clinical evaluation was conducted in accordance with the Declaration of Helsinki and approved by the relevant Institutional Review Boards. Umbilical cords from healthy donors (< 40 years) were collected with informed consent under the approval of the Institutional Review Board of Samsung Medical Center (IRB No. 2016-07-102-037, approved on 9 August 2022). The single-arm, open-label clinical protocol was reviewed and approved by the Institutional Review Board of the Korea Institute of Dermatological Sciences (IRB Nos. KIDSIRB-2024-1116, KIDSIRB-2024-1105, and KIDSIRB-2024-1124, all approved on 21 August 2024), an organization accredited by the Ministry of Food and Drug Safety of the Republic of Korea.”

• Institutional Animal Care and Use Committee (IACUC) Statement:
• “All animal procedures were approved by the Institutional Animal Care and Use Committee of Samsung Medical Center (IACUC No. 20230919001, approved on 16 October 2023)) and were performed at the Samsung Medical Center Preclinical Research Center in accordance with relevant guidelines and regulations.”

Comments 2: Conflict of Interest (COI) Statement [p.17, lines 532-535]

• Response 2: As per the editorial requirement, we have added the following statement before the Acknowledgments section:

“Author Oh Young Bang has been affiliated with S&E Bio since 2019, Author Na Eun Lee since 2022, and Author Ji-Eun Kim since 2023. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.”

Comments 3: Western Blot Raw Data Submission [p.16, lines 498-501]

• Response 3: We have uploaded the uncropped, untouched full original Western blot images corresponding to Figure 2 as Supplementary Figure S2, in compliance with the editorial request. For the reviewers’ convenience, we provide a ‘Raw Data Summary’ compressed archive that compiles the complete raw data underlying all submitted figures

Supplementary Materials: “The following supporting information can be downloaded at: https://www.mdpi.com/article/doi/s1, Figure S1: Localization of DiI-labeled WJ-MSC secretome coated spicules in mouse skin, Figure S2: Uncropped, full-length Western blot images corresponding to Figure 2.”

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

We appreciate the reviewer’s positive evaluation

Are all the cited references relevant to the research?

Yes

Thank you for confirming that the cited references are appropriate.

Is the research design appropriate?

Can be improved

We agree with the reviewer’s comment.

Are the methods adequately described?

Yes

We are grateful that the reviewer found our methodology adequately described.

Are the results clearly presented?

Can be improved

We thank the reviewer for this comment. To further enhance clarity, we have specified the sample size (N) in the figure legends and ensured that all statistical analyses are clearly described. We believe that these additions improve the transparency of the presented results without altering the main findings.

Are the conclusions supported by the results?

Can be improved

We appreciate the reviewer’s feedback. We carefully re-checked the Discussion and Conclusion sections and confirmed that they are consistent with the presented data. To strengthen this alignment, we have made minor refinements in wording to explicitly highlight how the key findings support our conclusions, without changing the overall interpretation of the study.

3. Point-by-point response to Comments and Suggestions for Authors

Comment 1: Figure 2 Effects of WJ-MSC secretome on skin cell function; Replicates/statistics not specified : The legend says “mean ± SEM” but does not indicate n (how many biological replicates or independent experiments). Western blot presentation ;No molecular weight markers shown, The loading control (e.g., β-actin) is not visible in the figure, Quantification method (densitometry normalization) is not described in legend. Statistical test : The figure legend lacks the specific test used (only p-value symbols shown). Suggestion: Include uncropped blots in supplementary data, add loading controls, and indicate exact sample size/test.

Ÿ   Response 1: Thank you for invaluable comments. We have uploaded the uncropped, untouched full original Western blot images corresponding to Figure 2 as Supplementary Figure S2. For the reviewers’ convenience, we provide a ‘Raw Data Summary’ compressed archive that compiles the complete raw data underlying all submitted figures.

Ÿ   Figure 2. Effects of WJ-MSC secretome on skin cell function. (A) Proliferation of fibroblasts (HS68) and keratinocytes (HaCaT) treated with WJ-MSC secretome (0-3.9 mg/mL) for 24 h (n = 5 independent replicates per group). (B) Scratch wound healing of HaCaT cells after 18 h treatment with WJ-MSC secretome (n= 4 independent replicates per group). (C) UVB-irradiated fibroblasts (30 mJ/cm²) showing restored collagen (COL1A1,180~200kDa; COL3A1,110/140kDa) expression upon WJ-MSC secretome treatment. β-actin(43kDa) was used as a loading control; uncropped blots are provided in Supplementary Figure S2. TGF-β served as a positive control (n = 3 independent experiments). (D) Type I procollagen secretion in fibroblasts following WJ-MSC secretome treatment (n= 5 independent replicates). (E) Antioxidant activity assessed by Trolox equivalent antioxidant capacity (TEAC) assay (n = 5 independent replicates). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Comment 2: Figure 4. Skin penetration of secretome-coated spicules (mouse); DiI is prone to false-positive labeling (it can diffuse). Authors should clarify how they confirmed that signal represents secretome, not free dye.

Ÿ   Response 2: We appreciate the reviewer’s insightful comment regarding the potential false-positive labeling associated with DiI. To ensure that the fluorescence signal represented secretome-associated particles rather than free DiI, the following precautions and validation steps were taken:

Ÿ   Rigorous removal of unbound dye: After labeling, the DiI-stained secretome was purified by sequential ultrafiltration (100 kDa MWCO) followed by phosphate-buffered saline (PBS) washing steps (3×) to completely remove free dye. The final filtrate was visually inspected to confirm the absence of residual fluorescence.

Ÿ   Negative control validation: As a control, DiI solution without secretome was subjected to the same coating and washing procedures and applied to the spicules. No detectable fluorescence was observed in this control, confirming that the signals in Figure 4 originated from the secretome-associated DiI.

Ÿ   Co-localization confirmation: The fluorescence distribution pattern of DiI-labeled secretome coincided with the penetration path of spicules, supporting that the signal corresponded to secretome localized within microchannels rather than diffused dye.

Ÿ   These additional details have been included in the revised Methods (Section 4.10. 13p, line 395-14p line 404) and mentioned in the Figure 4 legend for clarity.

Comment 3: Figure 5. Enhanced skin absorption and skin parameter improvements (clinical); Device labeling error :The figure legend says “3D Raman spectroscopy (VivaScope, Germany)”. VivaScope is confocal reflectance microscopy, not Raman. This is a serious mislabeling of the imaging technique.

Ÿ   Response 3: [15p line 444/Figure5] We sincerely thank the reviewer for pointing out this important labeling error. The instrument used was indeed confocal reflectance microscopy (VivaScope, Germany), not Raman spectroscopy. The figure legend has been corrected accordingly. Thank you for your careful review and valuable feedback.

Comment 4: Figure 6 .Reduction of wrinkles and pigmentation (clinical, 2 weeks); Missing raw values: Shows only % reduction, no absolute wrinkle index or pigmentation area values. Issue: No responder data: Does not show variability across subjects (e.g., whether all improved or just a subset). Suggestion: Add table of absolute values, show variability (e.g., box plot), and clarify blinding in methods/legend.

Ÿ   Response 4: We appreciate the reviewer’s comment. The variability across individual participants has been visualized using dot plots to represent each subject’s raw data points. This format clearly indicates inter-individual variation and responder distribution.

4. Response to Comments on the Quality of English Language

We appreciate the reviewer’s comment regarding language. Accordingly, the manuscript has undergone thorough English‑language editing by a native English‑speaking editor, and we have incorporated the revisions throughout the text.

[1p, Title]

Point 1: Nano-Encapsulated Spicule Delivery Enhances Deliver of Wharton’s Jelly MSC Secretome and Skin Rejuvenation in Preclinical and Clinical Evaluation

Response 1 Nano-Encapsulated Spicule Delivery Enhances Delivery of Wharton’s Jelly MSC Secretome and Skin Rejuvenation in Preclinical and Clinical Evaluation

[2p, line 39]

Point 2: “…extrinsic stressors, such as ultraviolet (UV) irradiation, oxidative stress, and environmental pollutants, accelerate the degenerative changes [1].”

Response 2 : “…extrinsic stressors, such as ultraviolet (UV) irradiation, oxidative stress, and environmental pollutants, accelerate degenerative changes [1].”

[2p, line 68]

Point 3: “…enabling efficient penetration of therapeutic agents with favorable safety profiles.”

Response 3 “…enabling efficient penetration of therapeutic agents with a favorable safety profile.”

[12p, line 332-333]

Point 4: “Adipogenesis was visualized by lipid droplet accumulation using Oil Red O staining... Osteogenesis was confirmed by calcium deposition detected with Alizarin Red S staining... Chondrogenesis was assessed by sulfated glycosaminoglycan accumulation using Alcian Blue staining.”

Response 4 “Adipogenesis was visualized by Oil Red O staining, osteogenesis by Alizarin Red S staining, and chondrogenesis by Alcian Blue staining.”

[5p line 144- 145]

Point 5: “…roughened surface with nanoparticulate deposits on coated spicules, consistent with deposition of secretome-derived nanocapsules (Figure 3B).

Response 5 “…roughened surface with nanoparticulate deposits on coated ones, suggestive of secretome deposition (Figure 3B).”

[6p, line 170-172]

Point 6:“…Quantification confirmed greater penetration in the secretome-coated spicule group versus control (p = 0.0152) and versus secretome alone (p = 0.0096), with no difference between control and secretome alone (ns, p = 0.9050).”

Response 6“…Quantitative analysis confirmed that secretome-coated spicules achieved significantly greater penetration than either control (p = 0.0152) or secretome alone (p = 0.0096), whereas the secretome-alone group did not differ from the control (ns, p = 0.9050)”

[9p, line 203]

Point 7: “3D Raman spectroscopy (VivaScope, Germany)”

Response 7 “3D confocal reflectance microscopy (VivaScope, Germany)”

[14p line 401]

Point 8:“50 μL of DiI-labeled samples was topically applied”

Response 8 “50 μL of the DiI‑labeled preparation”

[9p line 211-214]

Point 9: Figure 6 (Wrinkle & Pigmentation) “Data are presented as mean ± SEM; **p < 0.01, **p < 0.001.”

Response 9: “Data are presented as mean ± SEM (n = 21 participants). Absolute values for wrinkle index and pigmented area are provided in Supplementary Tables S1–S2. Statistical analyses were performed using paired t‑test or Wilcoxon signed‑rank test, as appropriate (two‑tailed); **p < 0.01; ***p < 0.001.”

5. Additional clarifications

The Discussion section has been extensively revised to address the reviewers’ comments. Specifically, we have (i) added appropriate references and figure citations for the key findings on transdermal penetration and clinical outcomes; (ii) corrected the oversimplified statement on skin permeability by citing authoritative sources; (iii) clarified the interpretation of SEM data and the novelty claim regarding spray-coated spicules; (iv) provided supporting citations for dosing, coating parameters, and encapsulation rationale; (v) added discussion of DiI labeling limitations and the need for orthogonal validation methods; and (vi) expanded the methodological limitations and future directions with appropriate literature on dosing optimization, heterogeneity, and clinical trial design. Collectively, these revisions improve scientific accuracy, citation integrity, and conceptual clarity throughout the Discussion.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. The detailed responses, along with the corresponding revisions and corrections, are highlighted below.

On behalf of all authors, we would like to sincerely thank you and the reviewers for the constructive comments and valuable suggestions on our manuscript entitled “Nano-Encapsulated Spicule Delivery Enhances Delivery of Wharton’s Jelly MSC Secretome and Skin Rejuvenation in Preclinical and Clinical Evaluation” (Manuscript ID: ijms-3914486), submitted to the International Journal of Molecular Sciences (Special Issue: Roles and Function of Extracellular Vesicles in Diseases: 3rd Edition).

We have carefully revised the manuscript according to the reviewers’ and editorial office’s instructions.

A detailed, point-by-point response to the reviewers’ comments is provided in the attached response file.

Comments 1: Institutional Review Board (IRB) Approval Dates

•  Response 1: [p.17, lines 501 and 503]
• We have added the approval dates for the IRB numbers as requested
• Revised text (Institutional Review Board Statement):

“This study involving human tissue and clinical evaluation was conducted in accordance with the Declaration of Helsinki and approved by the relevant Institutional Review Boards. Umbilical cords from healthy donors (< 40 years) were collected with informed consent under

the approval of the Institutional Review Board of Samsung Medical Center (IRB No. 2016-07-102-037, approved on 9 August 2022). The single-arm, open-label clinical protocol was reviewed and approved by the Institutional Review Board of the Korea Institute of Dermatological Sciences (IRB Nos. KIDSIRB-2024-1116, KIDSIRB-2024-1105, and KIDSIRB-2024-1124, all approved on 21 August 2024), an organization accredited by the Ministry of Food and Drug Safety of the Republic of Korea.”

• Institutional Animal Care and Use Committee (IACUC) Statement:
• “All animal procedures were approved by the Institutional Animal Care and Use Committee of Samsung Medical Center (IACUC No. 20230919001) and were performed at the Samsung Medical Center Preclinical Research Center in accordance with relevant guidelines and regulations.”

Comments 2: Conflict of Interest (COI) Statement [p.17, lines 517-520]

• Response 2: As per the editorial requirement, we have added the following statement before the Acknowledgments section:

“Author Oh Young Bang has been affiliated with S&E Bio since 2021, Author Na Eun Lee since 2022, and Author Ji-Eun Kim since 2023. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.”

Comments 3: Western Blot Raw Data Submission [p.16, lines 483-486]

• Response 3: We have uploaded the uncropped, untouched full original Western blot images corresponding to Figure 2 as Supplementary Figure S2, in compliance with the editorial request. For the reviewers’ convenience, we provide a ‘Raw Data Summary’ compressed archive that compiles the complete raw data underlying all submitted figures

Supplementary Materials: “The following supporting information can be downloaded at: https://www.mdpi.com/article/doi/s1, Figure S1: Localization of DiI-labeled WJ-MSC secretome coated spicules in mouse skin, Figure S2: Uncropped, full-length Western blot images corresponding to Figure 2.”

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

We appreciate the positive evaluation.

Are all the cited references relevant to the research?

Yes

Thank you for confirming the relevance of the citations.

Is the research design appropriate?

Yes

We appreciate the reviewer’s assessment.

Are the methods adequately described?

Must be improved

We expanded the Methods (Sections 4.12-4.16) to report exact sample sizes (n) for each assay and for animal/clinical analyses, inclusion/exclusion criteria, randomization/blinding procedures where applicable, reagent/equipment details (models and key settings), and the full statistical workflow (tests, post‑hoc comparisons, software and versions). Figure legends were updated to include n and the specific statistical tests used. Uncropped blots and complete numerical datasets are provided in the Supplementary Materials (Supplementary Figures S1-S2; Tables S1-S2).

Are the results clearly presented?

Can be improved

We have reorganized and improved the presentation of the figures and tables in the Results section and corrected instrument terminology (VivaScope is now described as confocal reflectance microscopy). The figure legends specify the sample sizes (n) and the statistical tests used. To enhance transparency, we have added absolute clinical values and subject‑level variability (Supplementary Tables S1-S2).

Are the conclusions supported by the results?

YES

We appreciate the positive assessment.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Reviewer: Overall concern about Methods & Results clarity

Results section: “Please indicate in each of the figures how many independent experiments have been performed.”

   Response 1: We thank the reviewer for this important request. We have updated all figure legends to report the exact number of independent replicates (n) and the statistical tests used.

   Fig. 2A (HS68 & HaCaT proliferation, 24 h): n = 5 per group; one‑way ANOVA with Tukey’s test.

   Fig. 2B (HaCaT scratch assay, 18 h): n = 4 per group; one‑way ANOVA with Tukey’s test.

   Fig. 2C (UVB fibroblasts WB, COL1A1/COL3A1): n = 3 independent experiments; densitometry normalized to β‑actin; one‑way ANOVA with Tukey’s test.

   Fig. 2D (Type I procollagen ELISA): n = 5; one‑way ANOVA with Tukey’s test.

   Fig. 2E (TEAC antioxidant assay): n = 5; one‑way ANOVA with Tukey’s test.

   Fig. 4B (mouse skin penetration, DiI): n = 3 mice per group; one‑way ANOVA with Tukey’s test; dye‑only control added in Supplementary Figure S1.

Comments 2: Section 2.1 & 2.2: Samples/Donors and scope

“How many different samples of WJ‑MSC were used?

• Response 2: “In response to the reviewer’s comment, we would like to clarify that Section 4.1 (WJ-MSC Master Cell Banking and Secretome Preparation) specifies that umbilical cords from three independent healthy donors (< 40 years) were collected with informed consent under IRB approval (Samsung Medical Center, IRB No. 2016-07-102-037, approved on 9 August 2022).”

Comments 3: “Characterization of the factors contained in the secretome‑coated spicules is necessary.”

• Response 3: We appreciate this important suggestion. In the current study, we performed multi‑modal characterization of the input secretome/EVs using antibody arrays (Fig. 1C), TEM and NTA (Fig. 1D-E), and EV surface markers by MACSPlex (Fig. 1F). Following coating onto spicules, we documented the nanoscale surface features by SEM (Fig. 3), and then evaluated functional readouts enhanced skin penetration in vivo (Fig. 4) and clinical improvements (Figs. 5-6). Together, these orthogonal datasets provide convergent evidence that the coated spicules carry bioactive secretome/EV components and deliver measurable biological effects.
• Direct quantitative profiling of bound factors on the spicule surface (e.g., elution followed by targeted immunoassays or proteomics) would require destructive processing of the coating layer and was therefore beyond the scope of the present work. We have clarified this point in the manuscript and explicitly acknowledge it as a limitation, while outlining targeted assays as a planned avenue for follow‑up validation. To aid transparency and reproducibility, we also added cross‑references in the text linking the pre‑coating characterization (Fig. 1C-F), the coated‑spicule morphology (Fig. 3), and the downstream functional outcomes (Figs. 4-6), and we provide the complete underlying datasets in the Supplementary Materials.

Comments 4: Figure 1C: “What is the control used here for the secretome analysis?”

• Response 4: We sincerely thank the reviewer for requesting clarification. For the antibody array shown in Figure 1C, the control was unconditioned culture medium processed identically to the secretome (no cells). Array signals were background-corrected, normalized to the internal negative-control spots, and reported as fold-change relative to the processed unconditioned medium. To minimize batch-to-batch variability and to ensure sufficient analyte for the screen, conditioned media from three independent donors were pooled and analyzed on a single array as an exploratory, semi-quantitative profiling experiment. The detailed methodology has been specified in Section 4.1 (WJ-MSC Master Cell Banking and Secretome Preparation; p.11, line 302-306).

Comments 5: In section 2.2.3. Collagen production was assessed by Western blotting. How many independent experiments were used to quantify the COL1A1 and COL3A1?

   Response 5: We appreciate the reviewer’s question. For Western blot quantification of COL1A1 and COL3A1, we performed n = 3 independent biological experiments, with densitometry normalized to β‑actin; uncropped blots are provided in Supplementary Figure S2. In a complementary assay, Type I procollagen in culture supernatants was quantified by ELISA with n = 5 independent replicates. These replicate numbers are now specified in the Figure 2 legend and Methods (4p, line 128 -132), and the raw values are available in the Supplementary materials (raw data summary).

Comments 6: In point 2.2.4. Antioxidant capacity, please add a brief comment on the meaning of this result.

   Response 6: Thank you for the suggestion. We added a brief interpretation: the TEAC assay showed a dose‑dependent increase in antioxidant capacity, indicating enhanced ROS‑scavenging potential of the WJ‑MSC secretome. This activity is mechanistically relevant to oxidative stress-driven skin aging and is consistent with the observed increases in procollagen/collagen (Fig. 2D-E).

   We have revised the section [2.2.4. Antioxidant capacity] (page 4, lines 136-137) to include the requested details.

Comments 7: Figure 4B and Supplementary Figure S1: It would be very helpful and much more interesting if you could present photos showing both the epidermis and the dermis from the same field of fluorescence.

   Response 7: We thank the reviewer for this helpful suggestion. The confocal micrographs we present already capture both the epidermis and the superficial dermis within the same field of view. To make this explicit, we used identical acquisition settings and scale bars across groups. Supplementary Figure S1 also includes representative images in which secretome‑coated spicules are visible within the epidermis (brightfield/reflectance channel, where available) together with DiI‑positive signal, as well as dye‑only controls processed in parallel. Taken together with the quantitative analysis in Figure 4B, these additions support the interpretation that the observed fluorescence originates from secretome carried by the coated spicules, rather than from dye‑related artifacts.

Comments 8: 4.1. WJ-MSC Master Cell Banking and Secretome Preparation: Adherent MSCs were expanded in MEM supplemented with 15% FBS. Please, add the exact composition of MEM media, i.e. glucose concentration, the presence or absence of nucleosides, and the use of L-glutamine or glutamax.

   Response 8: We appreciate the reviewer’s request for the exact medium composition. We used α‑MEM with nucleosides (Gibco, Cat. No. 12571‑063) formulated with L‑glutamine (2.0 mM; 292 mg/L), D‑glucose (1.0 g/L; 5.56 mM), sodium pyruvate (1.0 mM; 110 mg/L), and ribo‑ and deoxyribonucleosides; the formulation contains Earle’s salts and ascorbic acid and does not contain phenol red [11p, line 286-289].”

Comments 9: 4.6. Cell Proliferation and Viability: HS68 fibroblasts and HaCaT keratinocytes were seeded in 96-well plates at a density of 3 × 10³ cells/well and treated with various concentrations of WJ-MSC secretome for 24 h. Please, add the WJ-MSC secretome concentration at the M&M section.

   Response 9: [13p, line 329-331] We appreciate the reviewer’s suggestion. We have now specified the exact concentrations in Section 4.6 (Cell Proliferation and Viability) as follows: HS68 and HaCaT cells were treated for 24 h with WJ‑MSC secretome at 0, 1.6, 2.0, 2.8, and 3.9 mg/mL (protein‑equivalent), with the medium‑only condition serving as the 0 mg/mL control. This change has been incorporated into the Methods.

Comments 10: 4.8. Collagen and Antioxidant Assays: Fibroblasts were irradiated with UVB (30 mJ/cm²) and subsequently treated with WJ-MSC secretome. Please, include the data of how long did you irradiated the fibroblast, and what is the concentration of WJ-MSC secretome.

   Response 10: [13p, line 351-355] We appreciate the reviewer’s comment. UVB exposure in our study was defined by fluence (30 mJ/cm²) rather than by a fixed time. The exposure duration was calculated from the measured irradiance at the plate surface (I, mW/cm²) using t (s) = dose (mJ/cm²) / irradiance (mW/cm²); in our setup, the irradiance was [I] mW/cm², yielding an exposure time of [t] s to deliver 30 mJ/cm². [“UVB was delivered at a fluence of 30 mJ/cm² using a PCL-3000 UVB irradiation system (Boteck, Korea), with the plate positioned approximately 15 cm from the light source. Irradiance at the well surface was measured using the device’s integrated radiometer, and the exposure time required to deliver 30 mJ/cm² was approximately 10 s. During irradiation, the culture medium was replaced with PBS, and the plates were uncovered (lids removed) to avoid UV attenuation; cells were immediately returned to complete medium thereafter.”]

Comments 11: 4.9. Nano-Coated Spicules: Please add details of the amount of secretome and spicules used in the coating procedure. Also, how was the coating procedure carried out? Please, explain.

   Response 11: [13p. line 372-374] We appreciate the reviewer’s request for clarification. Details of the coating procedure have now been added to Section 4.9. Briefly, marine sponge-derived spicules (approximately 2.75 × 10⁵ spicules per preparation) were coated with WJ-MSC secretome (equivalent to 200 µg total protein) using a spray-coating method. The secretome suspension was atomized onto the spicules under gentle rotation (150 rpm) to ensure uniform coating, followed by air-drying at room temperature for 1 h under sterile conditions. The coated spicules were subsequently stored in airtight containers at room temperature until use. These details are now described in the Methods to improve reproducibility.

Comments 12: 4.10. In Vivo Skin Penetration Assay: Pleas, add the protocol used to label with DiI the spicules coated with WJ-MSC secretome. What os the amount of spicules coated with WJ-MSC secretome contained in the 50 μL of DiI-labeled samples?

   Response 12: [13p, line 380- 14p, line 389] We appreciate the reviewer’s request for clarification. The DiI labeling protocol and quantitative details have been added to Section 4.10. Briefly, WJ-MSC secretome–coated spicules (~2.75 × 10⁵ spicules per batch) were labeled with DiI (5 µM, Invitrogen, USA) in PBS for 20 min at room temperature in the dark, followed by two washes with PBS to remove unbound dye. The labeled preparation was resuspended in 50 µL PBS, corresponding to approximately 2.75 × 10⁵ coated spicules containing 200 µg protein-equivalent secretome, and applied topically to the dorsal skin. These details have been incorporated into the Methods to ensure reproducibility.

Comments 13: 4.11. Formulation of Spicule-Based Essence: What is the emulsifier used? What is the oil phase composed of? Please explain this protocol so that other researchers can replicate it.

  Response 13: [14p, line 394-403] The spicule-based essence was prepared using a two-phase oil-in-water (O/W) emulsification process. The aqueous phase contained the emulsifier (polyglyceryl-10 stearate, 2% w/w) and humectant (glycerin, 5% w/w) dissolved in purified water, while the oil phase consisted of caprylic/capric triglyceride (6% w/w) and squalane (3% w/w). Both phases were heated separately to 80 °C; the aqueous phase was homogenized at 6,000 rpm for 10 min, and the two phases were then combined under continuous stirring to form a uniform emulsion. The mixture was cooled to below 40 °C, at which point nano-coated spicules (2.75 × 10⁵ per preparation, coated with WJ-MSC secretome) were incorporated under aseptic conditions. The final formulation was filled into sterile airless containers and stored at ambient temperature until use.

Comments 14: 4.12. Study Design and Participants: 21 healthy adult female participants aged 34-66 years were included in the study. Were any inclusion and exclusion criteria applied in this clinical trial? They need to be explained.

   Response 14: We thank the reviewer for this valuable comment. The inclusion and exclusion criteria have now been described in Section 4.12 (Study Design and Participants). Briefly, participants were healthy adult females aged 34-66 years with mild-to-moderate facial wrinkles or pigmentation and no active dermatologic disease. Exclusion criteria included pregnancy or lactation, ongoing dermatologic treatment, history of cosmetic procedures or laser therapy within the past 3-6 months, use of topical retinoids within the past 4 weeks, and known hypersensitivity to any component of the formulation. These details have been added to ensure clarity and reproducibility.