A Multifunctional Peptide Linker Stably Anchors to Silica Spicules and Enables MMP-Responsive Release of Diverse Bioactive Cargos

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper presents the development of a multifunctional peptide linker designed to anchor strongly to silica spicules for transdermal delivery while enabling MMP-responsive release of bioactive cargos. Through screening a 180-member peptide library, the authors identified P176 as a lead candidate exhibiting high silica affinity, amphipathic helical structure, and robust binding characterized by biophysical methods and molecular dynamics simulations. The linker's G-P-H-C segment acts as an MMP-cleavable gate, demonstrating efficient kinetics and >90% cleavage within 60 min. Conjugation to diverse cargos—hydrophilic Vitamin C (87% yield) and hydrophobic Stigmasterol (77% yield)—followed by Franz diffusion assays showed MMP-dependent release (80–96% over 24 h) with minimal leakage, and released cargos retained bioactivity, enhancing collagen production in fibroblasts (~250% of control) and attenuating inflammation in macrophages. The novelty lies in engineering a single, sequence-programmed peptide that unifies wash-resistant noncovalent silica anchoring, built-in protease responsiveness, and modular compatibility with chemically disparate payloads under physiological conditions, overcoming limitations of traditional silane chemistries (hydrolytic instability, lack of responsiveness) and polydopamine coatings (batch variability, need for orthogonal degradable elements) for spicule-based regenerative delivery. However, there are a few issues that need to be addressed before suggesting for publication:

1. Could you specify the exact parameters for the molecular dynamics simulations, such as the force field version, integration timestep, ensemble type, and any restraints applied during equilibration?
2. To further improve the introduction, expand the discussion on existing linker strategies by incorporating examples of linkers from related fields to highlight the evolution toward controlled orientation and responsiveness such as doi.org/10.1021/jacs.5c17186
3. How was the helical content quantified from CD spectra (e.g., using specific algorithms like CDSSTR or CONTIN), and what assumptions were made about baseline corrections or buffer contributions?
4. In the conjugation assays, what criteria were used to confirm that the new HPLC peaks corresponded specifically to the desired conjugates rather than side products or aggregates?
5. To verify the binding affinity rankings, were independent replicates of the library screening performed with different spicule batches, and can you provide statistical measures (e.g., p-values) for the enrichment of V/V/K motifs?
6. For the MMP cleavage claims, were mass spectrometry fragmentation patterns analyzed to confirm the exact scissile bond in the G-P-H-C motif, and how do they align with known MMP substrate preferences?
7. Suggesting including direct comparisons with established silica linkers like APTES-glutaraldehyde or protein A-mediated immobilization to benchmark P176's performance in terms of binding density, release kinetics, and orientation control on spicules.
8. Discuss potential limitations in scalability, such as peptide synthesis costs or spicule sourcing variability, and propose strategies like sequence optimization for shorter motifs to enhance manufacturability.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents the development of a P176-based matrix metalloproteinase (MMP)-responsive cargo release platform. The authors conducted comprehensive physicochemical characterizations and in vitro biological evaluations to demonstrate P176’s silica-binding ability, MMP-responsive release behavior, and cargo release efficiency. However, the following issues should be addressed before publication.

 

1. The manuscript provides extensive data on P176’s MMP-specific cargo release capability but does not thoroughly discuss its enzyme selectivity (e.g.,potential hydrolysis by non-target enzymes). Including data on P176’s stability against irrelevant enzymes would strengthen the reliability of selecting P176 as a responsive moiety,providing more robust support for the clinical translation of this platform.

 

2. The manuscript states in the Methods section that statistical analyses such as t-test and one-way ANOVA were employed,with P<0.05 as the significance criterion. However,no specific statistical results (e.g.,p-values,F-values,degrees of freedom) are reported in the Results section,despite the term "significantly" being used twice to describe intergroup differences (Figures 5A and 5C). This disconnect between methods and results severely undermines the scientific rigor of the conclusions. The authors must supplement targeted statistical tests: label significance symbols (*,**) and corresponding p-values in relevant figures,specify test methods in figure legends,and include detailed statistical values in the text to ensure conclusions are evidence-based.

 

3. Figure 1 exhibits image presentation issues: P176 is not fully displayed in Figure 1A,and while the manuscript describes P176 as located in the upper right region of Figure 1B,the component is not clearly identifiable,and the image resolution is insufficient. The image quality should be optimized to ensure the target component is fully and clearly visualized as described,maintaining the accuracy of data visualization.

 

4. The frequency data of P015 in Figure 2E is inconsistent with the manuscript’s description (-15Hz). This discrepancy must be verified and corrected to ensure consistency between data and textual description.

 

5. Figure 4 has two irregularities: the bracket format in Panel A needs to be standardized to "()",and the data in Panel B is inconsistent with the textual description. The authors should verify and correct these issues one by one to ensure alignment between figure details and textual information.

 

6. Figure 6 has multiple presentation problems: Panels A and D lack scale bars; the caption formats of Panels B,C,E,and F are inconsistent. The missing scale bars should be added,and the annotation styles (e.g.,font,position,format) of all subpanels should be standardized per journal guidelines to enhance figure professionalism.

 

7. Figure legends are redundant and inconsistent,and the subpanel layout of Figure 6 (a multi-panel composite figure) is misaligned (uneven vertical spacing,unbalanced size ratios),which impairs visual coherence and readers’ comparative reading. It is recommended to simplify redundant legends (e.g.,delete duplicate legends for bar graphs with clearly labeled x-axes) and complete incomplete legends. Additionally,uniformly adjust the size,spacing,and annotation formats of all subpanels in Figure 6 to ensure a neat and standardized layout.

 

8. There is a significant discrepancy between the cell viability of the P176+MMP group in Figure 6C (approximately 150%) and the textual description (approximately 100%). The data accuracy should be verified and corrected to avoid compromising result credibility due to imprecise description.

 

9. References 3 (Muhammad,M.,et al.) and 6 (Corma,A.,et al.) lack complete page information. Accurate page ranges should be added to comply with academic citation standards and ensure literature traceability.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

All my concerns have been answered.