Review Reports

Comments 1: It was mentioned that the nano-formulations of NMN were prepared to address its low permeability. But Synerjet-assisted transdermal delivery can address this issue. Then why two different approaches together?

Response 1: We deeply appreciate the reviewer’s constructive comment, which allows us to clarify the critical biochemical and mechanical rationales behind our combinatorial approach. The decision to employ a nano-formulated NMN ampoule in tandem with the Synerjet system is based on three strategic pillars:

1) Minimizing Experimental Variables via Chemical Stabilization: As detailed in Table A2, the 10% nano-NMN ampoule contains other key bioactives (e.g., niacinamide and hyaluronic acid) with varying molecular weights and highly hydrophilic, labile properties. In their naive, unencapsulated state, these molecules are highly vulnerable to rapid environmental degradation and structural alterations. If naive NMN were used, it would be impossible to verify whether the delivered cargo remained structurally intact upon reaching the target layer. Therefore, nanoencapsulation was essential to preserve molecular integrity, eliminate degradation variables, and ensure that the effects mediated by the Synerjet system are biologically reliable.

2) Overcoming the Physical Limits of Nanoencapsulation Alone: Although nanoencapsulation protects volatile active ingredients, ample literature indicates that topically applied nanoparticles still face severe physical limitations in uniformly penetrating the lipophilic stratum corneum to reach the deep dermis. By integrating the Synerjet system, its electro-mechanical modalities (cold plasma and electroporation) safely bypass this physical barrier. This synergy allows the intact, stabilized nano-NMN particles to be effectively distributed into the target cutaneous matrix, unlocking the true therapeutic potential of the active cargo.

3) Complementary Synergy between a Mechanical Device and a Biochemical Cargo:

The Synerjet system is strictly a physical drug-delivery device; it cannot replicate or substitute the intrinsic biological actions of the therapeutic agents themselves. To achieve comprehensive skin rejuvenation, a highly advanced device must be paired with an equally optimized biochemical formulation. By establishing a clear comparison between the topical control and the combination test group, we successfully demonstrated that the Synerjet system serves as an ideal clinical accelerator that maximizes the innate, uncompromised efficacy of the nano-NMN formulation.

[Manuscript Revisions]: To explain these rationales clearly, we have updated the Introduction (Page 2, 61-88 and Discussion section (Page 13, 488-517) (formulation stability, barrier bypassing, and device-cargo synergy).

Comments 2: Under section 2.2 (Skincare Protocol: Sequential Cold Plasma and Electroporation), mention all the operating conditions, including the temperature. This will be useful for the readers and other investigators for reproducibility.

Response 2: We completely agree with the reviewer that detailed operational parameters are vital for scientific reproducibility. In accordance with your suggestion, we have explicitly detailed the environmental and device-specific conditions under Section 2.2.

1) Environmental Controls: All clinical procedures and subsequent instrumental measurements were conducted in a strictly regulated, climate-controlled facility devoid of direct sunlight or draft artifacts, maintaining a constant ambient temperature of 22 ± 2°C and a relative humidity of 50 ± 5%.

2) Device Operating Parameters: The cold plasma pretreatment was delivered in continuous mode via a single pass to prime the skin. Immediately afterward, a 2 mL dose of the nano-NMN 10% ampoule was administered via the microjet spray through a 10 mm EP tip-integrated nozzle. The concurrent EP function was locked at Level 1, operating at a frequency of 15 Hz and an electrical potential of 150 V.

[Manuscript Revisions]: The environmental environmental standards (22 ± 2°C,  a relative humidity 50 ± 5%) have been integrated into Section 2.2.1. The specific operating parameters (Level 1, 15 Hz, 150 V) under Section 2.2.2 have been highlighted (Page 3).

Comments 3: Subjects of a very wide range (age group) were selected. It is anticipated that the 40+ will have more of these issues, so why were subjects aged 30 included in this study?

Response 3: We completely agree with the reviewer’s excellent point that the evaluating efficacy of anti-aging and rejuvenation therapies is most clinically meaningful when conducted on a cohort presenting clear, established signs of skin aging. As the reviewer noted, our initial institutional review board (IRB)-approved recruitment protocol was broadly designed to accept candidates starting from age 30 to capture the very early stages of chronological aging by environmental stress such as UV radiation. While we cannot retroactively modify the official inclusion criteria established at the study's onset, we would like to clarify that the actual enrolled and analyzed cohort consisted strictly of middle-aged and elderly women ranging from 47 to 73 years old (Figure A1). Therefore, as the reviewer rightfully suggested, our data inherently reflects the therapeutic outcomes on advanced, well-established clinical signs of skin aging, free from the potentially confounding data of younger participants. We hope the reviewer finds this clarification reassuring regarding the clinical relevance of our study.

[Manuscript Revisions]: To ensure complete transparency and reflect the reviewer's valuable feedback, we revised age as 40 in Section 2.4 (Study Participants) on Page 4 that while the initial screening criteria accepted participants from age 30 (IRB protocol), the actual evaluated cohort was comprised exclusively of middle-aged and elderly individuals (47–73 years, Figure A1) with advanced skin aging.

Comments 4: What was the criterion for using four different depths (0.5, 1.5, 2.5, and 5.0 mm) in this study?

Response 4: We thank the reviewer for this insightful question regarding the parameters used during the assessment. We would like to clarify that the specific settings and threshold values were not arbitrarily selected; rather, they represent the pre-configured operating parameters natively programmed into the measurement system. Crucially, these device-set specifications are scientifically grounded in human skin anatomy and structural physiology. We hope this clarifies that the settings are inherently aligned with established skin structures:

The selection of the four discrete depths (0.5, 1.5, 2.5, and 5.0 mm) using the Moisture Meter D (Delfin Technologies) was intentionally designed to map the system's hydrating efficacy across distinct anatomical strata of the cutaneous and subcutaneous tissue.

1) 0.5 mm and 1.5 mm depths: These target the epidermis and upper papillary/reticular dermis. This is the zone where cold plasma and EP microcurrents directly alter permeability and where rapid cellular turnover takes place.

2) 2.5 mm depth: This captures the deep reticular dermis. Crucially, this depth showed the most profound divergence between the groups, with the Synerjet test group yielding a 188.02% higher moisture increment relative to the topical control (p < 0.001). This proves that the device creates a long-term "moisture reservoir" deep within the structural dermal matrix.

3) 5.0 mm depth: This represents the deep subcutaneous adipose tissue layer, which lies well beyond the therapeutic reach of cosmetic transdermal devices. As expected, neither group showed any significant deviation from baseline at 5.0 mm (p > 0.05). This depth served as an excellent internal negative control, validating that the device’s bio-stimulating and hydrating benefits are precisely concentrated within the upper 2.5 mm of regeneratively active skin tissue.

[Manuscript Revisions]: We have updated that this anatomically-based settings are pre-programed by manufacturer in Section 2.6.3 (Page 5, 182-186).  

Comments 5: Please verify the probe used for the skin elasticity measurement.

Response 5: We sincerely thank the reviewer for this verification request. We have re-checked our laboratory logs and device data sheets to confirm the instrument specifications utilized during the trial from Human Skin Clinical Trial Center Co., Ltd. [23]. Skin elasticity assessments were executed using the Cutometer® dual MPA 580 (Courage + Khazaka Electronic GmbH, Cologne, Germany) equipped with the standard 2 mm probe, which perfectly aligns with the statement in our manuscript. For the measurements, the device applied a software-controlled negative pressure cycle three times consecutively to deform the skin into the 2 mm probe aperture. The gross elasticity (R2) value was then calculated based on the tissue’s deformation and vertical recovery curves (as detailed in Table A3). We hope this clarifies the hardware setup used in our study.

[Manuscript Revisions]: Section 2.6.5 has been audited and remains tightly specified to maintain technical clarity (Page 5, 199-201).

Comments 6: Discussion of the data may be further improved.

Response 6: We sincerely appreciate the reviewer’s constructive suggestion to strengthen the discussion regarding our empirical findings. In accordance with this valuable feedback, we have significantly expanded the Discussion section by integrating recent scientific literature to solidify our results. Specifically, we have elaborated on the cellular mechanisms whereby NMN-induced NAD+ biosynthesis stimulates hyaluronic acid and collagen expression in dermal fibroblasts, which corroborates the deep hydration (TDC) profiles measured by the Moisturemeter D. Additionally, we have cross-referenced the delivery dynamics of our multi-functional platform with established transdermal drug-delivery studies to justify the structural improvements observed in advanced skin aging.

[Manuscript Revisions]

Please find the updated Discussion section on Pages 13–14 (518-560) and additional section 5. Limitations and Future Directions (Page 14, 561-580).

Comments 7: Give a separate section for the limitations of the work and how they can be addressed.

Response 7: We sincerely appreciate this constructive guidance. In accordance with the reviewer’s recommendation, we have significantly updated the final part of the Discussion section by extracting the limitations from the general text body and establishing a standalone subsection (Section 5. Limitations and Future Directions). This section clearly details the current constrains of our work alongside our specific mitigation strategies as below:

[Manuscript Revisions] Please find the updated Discussion section on Pages 13–14 (518-560) and additional section 5. Limitations and Future Directions (Page 14, 561-580).

5. Limitations and Future Directions

A primary limitation of this study is the structural divergence between the two inter-vention tracks—specifically, weekly clinic-based Synerjet treatments versus twice-daily at-home topical applications—which introduces potential confounding variables such as application frequency, setting, and professional administration. However, this design was intentionally selected as a real-world comparative effec-tiveness trial to reflect practical dermatological protocols; twice-daily clinic visits for device treatments are logistically unfeasible, while limiting topical ampoule use to once weekly would fail to represent a realistic, compliant skincare regimen. Addition-ally, this study is constrained by a relatively small sample size, the absence of histolog-ical or molecular validation of NAD+ upregulation, and the lack of a longer follow-up period to assess the durability of the observed improvements. Furthermore, the current protocol did not incorporate the Synerjet microjet function for intradermal delivery, which precluded the potential for further enhanced collagen remodeling and fully op-timized aesthetic outcomes for deep-seated target layers. Accordingly, future well-controlled investigations utilizing standardized application frequencies should address these limitations by expanding the mechanistic framework to include quanti-tative assessments of collagen synthesis rates, dermal matrix remodeling biomarkers, and the kinetics of skin regeneration following microjet-assisted NMN delivery. Such efforts will be essential for establishing the robust evidence base required for the for-mal dermatological endorsement of this combinatorial platform.

Comments 1: The Conflict of Interest section states "No conflicts to disclosure," yet two co-authors (S.-K. Park, M.-H. Kim) are employees of Hironic Co., Ltd., the manufacturer of the Synerjet device, and the study was funded by both Hironic and DEFY NUMBER Co., Ltd. (the product supplier). This is a serious omission that must be corrected with a full, transparent disclosure.

Response 1: We sincerely thank the reviewer for this critical and protective comment regarding the transparency of our study. We completely agree that full disclosure of any potential conflicts of interest is essential for maintaining scientific integrity, and we deeply apologize for any oversight or ambiguity in our initial submission.

To clarify the timeline and context of the authors' affiliations: the experimental phase of this study and the data collection were entirely conducted before the co-authors (S.-K. Park and M.-H. Kim) joined Hironic Co., Ltd. The manuscript was subsequently written after their employment in February 2026. Because the core research was executed independently prior to their corporate affiliation, we initially presumed it did not constitute a direct conflict; however, we fully recognize the reviewer's point that their current employment and institutional affiliations must be transparently declared to ensure complete objectivity.

We firmly believe that corporate affiliation should not preclude qualified researchers from contributing authorship, provided that full transparency is maintained, allowing the readers to make their own informed evaluations. Therefore, in strict accordance with the reviewer's valuable guidance, we have comprehensively updated the Conflict of Interest and Funding sections to provide a completely transparent and unrestricted disclosure. Furthermore, we would like to clarify a clerical error in the initial manuscript regarding the funding source. The original draft inadvertently included Hironic Co., Ltd. as a funding sponsor. We have thoroughly re-verified the administrative and financial records of this study and corrected this oversight. To be absolutely precise, this research was independently funded and conducted solely by DEFY NUMBER Co., Ltd.  Hironic Co., Ltd. did not provide any financial sponsorship or research grants for this work; their involvement is strictly limited to the current employment of the two co-authors as detailed above. We sincerely apologize for this initial misstatement and deeply appreciate the reviewer’s diligent review, which allowed us to ensure the accuracy of our institutional disclosures. 

[Manuscript Revisions]: Author Contributions, Informed Consent Statement, Conflicts of Interest, and Funding (Page 15, Lines 592-615) were revised as below:

○Author Contributions: Conceptualization, Resources, methodology, formal analysis, investigation, data curation, validation, writing—original draft preparation, supervision, visualization, project administration, funding acquisition, WH. Resources, project administration, supervision, JK, SN & JS. Conceptualization, methodology, validation, writing—review and editing, SKP. Conceptualization, writing—original draft preparation, visualization, MHK. All authors have read and agreed to the published version of the manuscript.

○Funding: This research was funded by DEFY NUMBER Co., Ltd.

○Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

○Conflicts of Interest: SKP and MHK are currently employed by Hironic Co., Ltd. However, the experimental phase and data collection of this study were completed entirely before their em-ployment. Hironic Co., Ltd. provided no financial support, research grants, or sponsorship for this study, which was independently funded and conducted solely by DEFY NUMBER Co., Ltd. The remaining authors declare no conflicts of interest.

Comments 2: The drafting assumes that synerjet is a known technology. It needs an introduction when it is inserted into the manuscript.

Response 2. We completely agree with the reviewer's valuable suggestion. Since the Synerjet system is an innovative transdermal delivery platform, providing a foundational introduction in the preliminary section will greatly improve the clarity and accessibility of our manuscript for the readers. In accordance with this guidance, we have integrated a dedicated paragraph in the Introduction (Page 2, Lines 66-82), Materials and Methods (Page 3, 98-132), and Discussion (Page 13, 499-517) that formally introduces the Synerjet technology, explicitly detailing its multi-functional mechanisms—specifically the sequential integration of atmospheric cold plasma treatment, electroporation (EP) cell-membrane permeabilization, and high-velocity microjet transdermal propulsion. We hope this addition contextualizes the technological rationale behind our study setup.

[Manuscript Revisions]: Please find Introduction (Page 2, Lines 66-82), Materials and Methods (Page 3, 98-132), and Discussion (Page 13, Lines 499-517).

Comments 3: The test group received clinic-based Synerjet treatment once weekly, while the control group self-applied the ampoule topically twice daily at home. This introduces multiple confounders (professional vs. self-application, clinical setting vs. home, frequency/dose differences).

Response 3: We sincerely appreciate the reviewer’s astute and methodologically rigorous comment. We completely acknowledge that the structural differences between the two groups—specifically, the clinic-based weekly Synerjet treatment versus the twice-daily at-home topical application—introduce distinct variables such as application frequency, setting, and professional administration. We would like to clarify that this study was designed as a real-world comparative effectiveness trial rather than a strict mechanistic vehicle-controlled study. In clinical dermatology practice, it is practically and financially unfeasible for patients to receive professional, high-velocity transdermal engine treatments twice daily in a clinic setting. Conversely, evaluating a weekly-only topical application at home would not reflect a realistic or compliant skincare regimen, as topical ampoules inherently require daily repetitive application to maintain baseline epidermal turnover support.

Therefore, our primary objective was to compare the practical clinical outcomes of two standard aesthetic tracks: a high-efficiency, device-assisted weekly professional protocol versus a conventional, patient-compliant daily home-care regimen. To address the reviewer's critical point and ensure complete transparency for the readers, we have added a dedicated paragraph acknowledging these operational confounders as an inherent study limitation in the newly established Limitations and Future Directions section (Page 14, Lines 562-569).

[Manuscript Revisions]: Please find 5. Limitations and Future Directions (Page 14, Lines 562-569.

Comments 4: How can the efficacy be attributed to NMN while other significant bioactive components (niacinamide, hyaluronates etc) are included in the formulation?

Response 4: We thank the reviewer for this highly critical and scientifically pertinent question. We agree that isolating a single active's efficacy in a multi-component formulation is challenging. However, we confidently attribute the pronounced deep-tissue improvements to the addition of NMN for the following reasons:

1) Direct NAD+ Biosynthesis: Unlike niacinamide, which is a distant precursor subject to strict enzymatic rate-limiting steps (via NAMPT), NMN acts as the immediate, direct precursor to NAD+. It bypasses these bottlenecks to rapidly elevate intracellular NAD+ levels in dermal fibroblasts.

2) Deep Dermal Hydration vs. Superficial Moisture: Topical hyaluronic acid primarily acts as a superficial humectant within the upper stratum corneum. In contrast, our MoistureMeter D data revealed highly significant increases in tissue dielectric constants (TDC) at deeper dermal layers 1.5 mm and 2.5 mm. This deep hydration profile cannot be achieved by topical hyaluronates alone and directly aligns with NMN’s capacity to stimulate intrinsic hyaluronic acid synthases HAS2 inside the dermis.

3) Formulation Synergy: While niacinamide and hyaluronates provide baseline epidermal support, the advanced anti-aging outcomes (e.g., structural pore reduction and micro-wrinkle attenuation) strongly correlate with the robust up-regulation of cellular energy metabolism unique to NMN-driven NAD+ amplification.

[Manuscript Revisions]: To ensure complete clarity and acknowledge the complex interplay of these ingredients, we have added a clarifying paragraph in the Discussion section (Page 13~14, Lines 518-548) to explicitly contextualize the distinct role of NMN relative to the other formulation components.

Comments 5: The study is entirely open-label (participants and investigators aware of group assignments). Given the subjective nature of some assessments and the self-reported satisfaction questionnaire, observer/participant bias cannot be excluded.

Response 5: We sincerely thank the reviewer for this crucial methodological critique. We completely agree that in an entirely open-label study design, the potential for participant and observer bias cannot be theoretically excluded, particularly regarding subjective metrics like self-reported satisfaction questionnaires. We would like to clarify that executing a strict double-blind setup was technically and operationally restricted due to the physical nature of the intervention; the Synerjet system involves distinct tactile sensations (high-velocity microjet spray, electrical stimulation from electroporation, and atmospheric plasma contact) that make it virtually impossible to blind the participants or the administering investigators without employing a sophisticated sham device. To systematically mitigate this inherent open-label limitation and isolate true therapeutic efficacy from psychological bias, our study intentionally relied on blinded data analysis and highly objective, hardware-driven diagnostic tools as our primary endpoints. All key dermatological parameters—such as deep skin hydration (measured via the tissue dielectric constant by Moisturemeter D) and structural laxity/pore volume—were quantified using automated, calibrated bio-engineering devices rather than clinician grading. Furthermore, to prevent observer bias during data processing, all instrumental datasets were anonymized and analyzed by an independent statistician who was completely blinded to the group assignments.

[Manuscript Revisions]: We fully accept the reviewer's point that self-reported data should be interpreted with caution. Consequently, we updated the discussion section (Page 14, Lines 549-560) to clearly discuss this open-label framework and how we controlled for potential bias.

Comments 6: The reported percentages (e.g., 170.56%) are relative improvement rates of the test group vs. the control group, not absolute efficacy values. These are prominently featured in the abstract and throughout the text without adequate clarification, creating an inflated impression of efficacy.

Response 6: We sincerely thank the reviewer for this crucial statistical and editorial guidance. We completely agree that presenting high percentage values (e.g., 170.56%) without explicitly defining them as relative improvement rates against the control group could inadvertently create an inflated impression of absolute efficacy. In strict accordance with the reviewer's recommendation, we have tried to revise the Abstract ~ Discussion to ensure complete transparency. We have changed terms like "efficacy rate" or "improvement" to "relative improvement rate compared to the control group". We would also like to clarify that the absolute baseline and post-treatment values (Mean ± SD) for all dermatological parameters are comprehensively detailed in our clinical report (see Reference 23). This ensures that readers can accurately evaluate both the absolute physiological changes and the relative therapeutic delta.

[Manuscript Revisions]: We changed terms like “improvement” to “relative improvement” across all pages.

Comments 7: The discussion extensively invokes NAD+ upregulation, collagen synthesis, melanosome transfer inhibition, and dermal remodeling, yet the study provides no molecular, histological, or biomarker data to support any of these claims. All endpoints are macroscopic/instrumental. The mechanistic narrative is speculative and should be substantially minimized.

Response 7: We completely agree with the reviewer's critical point. Since our endpoints are strictly macroscopic and instrumental, definitively claiming molecular pathways without direct histological or biomarker data can appear speculative. In accordance with your guidance, we have thoroughly revised the Discussion section to substantially tone down and minimize the mechanistic narrative through the following modifications (Page 13, 14).

[Manuscript Revisions]: We have revised Discussion section to tone down (Page 13-14, Lines 513-548)

Comments 8: Wonkyu Hong appears as both first and last author with the same affiliation (Class One Clinic).

Response 8: We appreciate the reviewer's diligent review. We would like to clarify that Wonkyu Hong is indeed both the primary investigator (first author) who executed this study and the corresponding author (last author) responsible for the overall scientific integrity of the manuscript. This dual role is intentional and accurately reflects his primary contribution to both the clinical execution and the conceptual design of this research.

Comments 9: A3 does not appear correctly

Response 9: We sincerely appreciate the reviewer’s diligent and meticulous review in pointing out the formatting issue with Table A3. Regarding this comment, we would like to inform the reviewer that we have already contacted the Cosmetics Editorial Office regarding the truncation and page-split layout issues of Table A3 (Schedule of Assessments) in the PDF proof. The journal's assistant editor has formally responded and assured us that this formatting glitch will be thoroughly addressed. Specifically, during the final production and typesetting stage following official acceptance, their specialized production team will automatically check and adjust the layout to ensure that all tables and figures are perfectly formatted, fully visible, and fully compliant with the journal's publication standards.

We are deeply grateful to the reviewer for bringing this to our attention with such careful oversight, which allowed us to proactively coordinate with the editorial team and ensure the visual quality of our manuscript.