From Bacterial Extract to Breakthrough Therapy: Pseudomonas fluorescens-Enabled Green Synthesis of pH-Responsive Chitosan–Silver Hybrid Nanoparticles for Next-Generation Pulmonary Drug Delivery Anti-MDR Treatment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors The manuscript is well written and organized.  However there are some sections that need to be improved/clarified:

• The method of HPLC analysis to quantify ciprofloxacin was not described (sample preparation and equipment setup). There is a time when reference 35 appears after an HPLC analysis description, however HPLC is not mentioned in such reference.
  
  
• The stimuli-responsive behavior of the CS–Ag HNPs is not hypothesized in detail. It is mentioned is due to the chitosan inherent capabilities, but more details should be given.
  
  
• Was there a formulation made in the absence of silver? If such formulation was prepared those results should be included in the manuscript, instead of a simple comparison with free ciprofloxacin. This concerned is raised due to the use of Ag in a pulmonary delivery system. It is well established that silver nanoparticles raise some concerns on their application in pulmonary drug delivery systems. Silver tends to be pro-inflammatory, can cause tissue damage and bioaccumulation, and even be considered as genotoxic. These potential drawback should also be addressed in the manuscript if no data is provided concerning a Ag-free formulation (there are multiple examples of recent publications revising this topic that should be used to address this drawback).

 

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, Alabbosh, Elmetwalli et al. describe the development of novel ciprofloxacin-chitosan-silver hybrid nanoparticles (HNPs) designed for pulmonary drug delivery. The physicochemical properties of the nanoparticles, including their hydrodynamic diameter and Zeta potential, were thoroughly characterized. The authors demonstrate that the HNPs exhibit superior antimicrobial activity against multidrug-resistant pathogens, achieving up to a fourfold reduction in the minimum inhibitory concentration (MIC) compared with free ciprofloxacin. Finally, the study proposes detailed synergistic mechanisms of action for the HNPs, involving biofilm disruption, reactive oxygen species (ROS) generation, and favourable biocompatibility with lung cells.

The manuscript is well written, and the results have been thoroughly discussed, but it contains important issues that must be addressed before it can be published.

• In section 3.2, it is stated that the bandwidth indicates the presence of polydisperse AgNPs in the CS matrix; however, the width of the absorption spectrum due to SPR depends on factors beyond polydispersity.
• A significant concern is that multiple complex analytical and mechanistic results presented in section 3.7 and discussed in detail lack corresponding description of the methods or techniques used. The method for ROS generation, membrane disruption, DNA inhibition, efflux inhibition, resistance mechanism bypass are not described.
• When the authors compare the antimicrobial activity of individual components to assess synergistic effects, they claim that the combined effect of CS-AgNPs and ciprofloxacin alone exceeds the 100% inhibition achieved by ciprofloxacin-CS-AgNPs. It could be possible to modify the experimental conditions to properly observe a true synergistic effect of the ciprofloxacin and CS-AgNPs combination by reducing the doses of both components.
• Regarding the human lung epithelial cell line used for biocompatibility testing, in methods and abstract section human airway epithelial cells Calu-3 is reported to be used, meanwhile in figure 8 caption the authors explicitly states that cell viability was evaluated using A549 cells.

 

• The table 3 compares the developed ciprofloxacin-CS-AgNPs with other formulations, but the references cited do not correspond to the formulations being discussed. Some of the bibliographic references throughout the text should be reviewed.
• The entire section 4, Molecular Docking Analysis and Binding Interactions, and figure 9 is presented without any accompanying methodology. The methods section fails to describe the software used, the protein and ligand structures or the parameters for the simulation. The amino acids involved in the interaction are identified in the figure, but the protein used in the study is not specified. Figure 9B displays binding energies under different pH conditions, but methodology used is not reported. The authors provide very limited explanation regarding the data plotted in the figure 9E, source of the data and methodology used to generate it. The results in this section are poorly described and discussed.
• Figures 10, 11 illustrate the complex proposed mechanisms of action, which are not fully supported by the explicit experimental methods. Figure 10 describes a detailed six-stage antimicrobial cascade. While the illustration is comprehensive, it incorporates specific outcomes that lack experimental confirmation, as quantifying membrane lipid peroxidation, protein denaturation, cellular ATP depletion. The figure, therefore, serve more as a conceptual model based on the known behaviour of chitosan and silver components rather than a visualization of data entirely generated within this study.
• Although the text boasts a 90% ROS generation efficiency, the methodology provides no means to confirm these specific mechanisms. The illustration of the biochemical effect of silver nanoparticle in Figure 11 is thus likely a theoretical representation, based on bibliography.
• Figure 12 illustrates the dual-action mechanism for biofilm disruption, including chitosan's role in enzymatic degradation of polysaccharide chains and AgNPs-mediated oxidative stress. However, the enzymatic degradation activity is not demonstrated in this work and remains speculative. Additionally, the mention of "alginate" in Figure 12 appears to be an error.

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

While the concept is promising and highly relevant, the study currently lacks mechanistic validation, in vivo data, and detailed compositional characterization to justify publication in Pharmaceutics.

1. How do the authors ensure batch-to-batch reproducibility of bacterial extracts for nanoparticle synthesis?
2. Could this green synthesis approach be generalized to other antibiotics beyond ciprofloxacin?
3. What is the long-term storage stability of the lyophilized HNPs (6 months, humidity tests)?
4. Did the authors perform any rheological or aerosol dispersion stability tests in inhalation devices?
5. Have in vivo pharmacokinetic or biodistribution studies been planned to confirm targeted lung delivery?
6. Add scale bars to TEM and CLSM images.
7. Figure 6 legends lack detail (staining methods, magnification).
8. The Discussion is excessively long and partly repetitive of Results. Please condense.
9. The Conclusion overstates clinical relevance; rephrase to emphasize potential, not proof.

 

Author Response

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Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have substantially improved the manuscript with new discussion, and they have completed the previously missing methodological information. However, Table 3 should be revised because it appears to be the same as in the original manuscript, and the references have not been properly corrected.

 

Author Response

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