Review Reports - Targeted Drug Delivery of Anticancer Agents Using C5N2 Substrate: Insights from Density Functional Theory

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work utilizes density functional theory (DFT) calculations to investigate the C₅N₂ carrier-based drug delivery mechanisms. The author made a detailed statement and explanation of the computational methods chosen for their study. Several physical parameters, such as bond length, interaction energy, reduced density gradient, and energy gap, were applied to characterize the interactions between drugs and the C₅N₂ substrate. The drug delivery mechanisms are further unveiled by electronic properties, recovery time, and solvent effect. While the study is methodologically sound and the results are well presented, the significance of the investigated drug delivery mechanisms and their potential application scenarios have not been sufficiently discussed. Therefore, I recommend this work for publication in Chemistry after a minor revision:

1. The authors claim that no findings have been reported regarding the drug-delivering capabilities of the C₅N₂ substrate. Please verify the accuracy of this statement, as some published studies have indicated that C₅N₂ nanoparticles possess potential for drug delivery applications:

[1] Angewandte Chemie International Edition, 2019, 58, 6290-6294.

[2] Particle & Particle Systems Characterization, 2021, 38, 2100193.

[3] Angewandte Chemie International Edition, 2021, 60, 16641-16648.

2. Please revise the abstract to improve readability by reducing the use of abbreviations. In its current form, the heavy use of abbreviations makes it difficult to read and follow.

3. The authors present several physical insights into the drug delivery mechanisms based on C₅N₂. Given that the employment of C₅N₂ as a drug delivery carrier has been seldom reported, the manuscript would benefit from a more comprehensive discussion of its advantages over other commonly used drug delivery materials. Furthermore, potential real-world application scenarios of the studied mechanisms should be elaborated upon, particularly after the presentation of the computational findings.

Author Response

Manuscript ID chemistry-3643418

Point-by-Point Response to the Reviewers

We are really thankful to the editor and the reviewers for sparing their precious time and providing us with valuable comments on our recently submitted manuscript to chemistry. Reviewers' comments were really helpful and encouraging for us in improving the quality of the manuscript. Point-by-point responses (given in blue text) are given for all comments (in black text).

Reviewer # 1

The authors claim that no findings have been reported regarding the drug-delivering capabilities of the C₅N₂ Please verify the accuracy of this statement, as some published studies have indicated that C₅N₂ nanoparticles possess potential for drug delivery applications:
[1] Angewandte Chemie International Edition, 2019, 58, 6290-6294.
[2] Particle & Particle Systems Characterization, 2021, 38, 2100193.
[3] Angewandte Chemie International Edition, 2021, 60, 16641-16648.
We thank the reviewer for bringing this to our attention. We agree with the comment and have revised the manuscript accordingly to reflect the previously reported drug delivery potential of C₅N₂ nanoparticles. The sentence has been corrected and the suggested references have been cited in the Introduction (Page 3, Line 117-118).
Please revise the abstract to improve readability by reducing the use of abbreviations. In its current form, the heavy use of abbreviations makes it difficult to read and follow.

We acknowledge that the original abstract included numerous abbreviations, which could hinder readability for a broader audience. To address this, we have thoroughly revised the abstract to reduce the use of abbreviations and enhance clarity. Most technical terms have been written in full, and the structure has been improved for better flow.

The authors present several physical insights into the drug delivery mechanisms based on C₅N₂. Given that the employment of C₅N₂ as a drug delivery carrier has been seldom reported, the manuscript would benefit from a more comprehensive discussion of its advantages over other commonly used drug delivery materials. Furthermore, potential real-world application scenarios of the studied mechanisms should be elaborated upon, particularly after the presentation of the computational findings.

We appreciate the reviewer’s insightful suggestion. In response, we have added a detailed discussion comparing the advantages of C₅N₂ with those of other commonly used drug delivery materials (see the discussion section, Page 3, lines 104-122) and elaborated on potential real-world applications based on our computational findings. (see discussion section Page 21- Line 714-726).

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors presented a study regarding the employs a C5N2-based targeted drug carrier to study the delivery mechanism of anticancer drugs, particularly cisplatin, carmustine, and mechlorethamine, using density functional theory. The manuscript deserves to publish in Chemistry after a minor correction. I would like to suggest introducing changes before publishing in Chemicals.

The Authors should revise in the manuscript as the following points:

I suggest changing the title. The name C5N2 is not clear to me. If the Authors want to use such an abbreviation, it is worth introducing it first. Without an introduction, it is not clear what substance it is.
Why did the authors choose such a functional and a ductional basis for DFT calculations? Are there any comparisons with experiment?
Abstract, line 24: it should be: “Electron Localization Function (ELF)…”
Why The authors performed calculations in a gas environment? I would suggest choosing and testing several solvents.
Place the atom labels on the drawing.
Table 2: the markings are wrong. For me G is Gibbs Free energy. H is also incorrect.
Please complete conclusions for future experimental work.

Author Response

Manuscript ID chemistry-3643418

Point-by-Point Response to the Reviewers

Reviewer # 2

The Authors presented a study regarding the employs a C₅N₂-based targeted drug carrier to study the delivery mechanism of anticancer drugs, particularly cisplatin, carmustine, and mechlorethamine, using density functional theory. The manuscript deserves to publish in Chemistry after a minor correction. I would like to suggest introducing changes before publishing in Chemicals. The Authors should revise in the manuscript as the following points:

I suggest changing the title. The name C₅N₂ is not clear to me. If the Authors want to use such an abbreviation, it is worth introducing it first. Without an introduction, it is not clear what substance it is.
We have introduced the abbreviation "C₅N₂" now clearly in the Introduction (Page 3, line 98-100), describing it as a nitrogen-rich covalent carbon nitride with a 5:2 carbon-to-nitrogen ratio.
Why did the authors choose such a functional and a ductional basis for DFT calculations? Are there any comparisons with experiment?

We appreciate the reviewer’s insightful question. We selected the PBE0-D3BJ functional combined with the def2-SVP basis set because this level of theory has been extensively validated for accurately describing noncovalent interactions, which are the core of our drug@C₅N₂ analysis. The PBE0 hybrid functional balances exchange and correlation effects well, while the D3BJ dispersion correction reliably captures van der Waals and hydrogen-bonding interactions which are equally critical in drug adsorption systems. Literature benchmarks confirm that PBE0-D3BJ/def2-SVP produces reliable geometries and energetics, especially in systems involving π–π stacking and H-bonding (e.g., comparison with benzene‐graphitic and drug–carbon-nitride systems) [1, 2] (We added a discussion to the manuscript, see page 4, lines 162-167)

Although our study is purely computational, the interaction energy trends are aligned well with those reported in experimental and combined experimental–theoretical studies of similar carbon nitride and carbon-based nanocarriers. For example, aspirin showed stronger adsorption on carbon nitride nanotubes than on pure carbon nanotubes (0.67 eV vs. 0.51 eV) using comparable DFT models [3]. Similarly, molecular dynamics and DFT simulations of doxorubicin on carbon nitride nanosheets have shown consistent noncovalent interaction trends [4]. These observations are in good agreement with our computed interaction energy order for the cisplatin, carmustine, and mechlorethamine-based systems, lending support to the validity of our computational approach. (See Page 21, Line 726 -728)

Abstract, line 24: it should be: “Electron Localization Function (ELF)

The term has been corrected to “Electron Localization Function (ELF)” in the abstract as suggested.

Why The authors performed calculations in a gas environment? I would suggest choosing and testing several solvents

While we initially performed calculations in the gas phase to obtain baseline interaction energies and electronic properties under ideal conditions, we also conducted calculations in the presence of water as a solvent using the SMD model (See Section 5 - Solvent Effect). Water was specifically chosen because it is the most relevant biological solvent and the main component of human body fluids. This allows for a more realistic simulation of the drug@C₅N₂ interactions under physiological conditions [5]. The solvent-phase results (e.g., solvation energy, dipole moment, and electronic properties) are now discussed in more detail in the revised manuscript (See Section 5 - Solvent Effect).

Place the atom labels on the drawing.

To maintain the clarity and visual quality of the figure, we have provided detailed atom labeling and descriptions in the figure caption and in the main text to ensure clear identification of all relevant atoms. (See Figure 1, Page 7)

Table 2: the markings are wrong. For me G is Gibbs Free energy. H is also incorrect

We appreciate the reviewer’s observation regarding the notation in Table 2. In this context, we would like to clarify that G(r) and H(r) do not refer to Gibbs free energy or enthalpy, but rather follow the conventional quantum chemical topological analysis definitions:

G(r) denotes the kinetic energy density, V(r) denotes the potential energy density

H(r) = G(r) + V(r) denotes the total energy density

This notation is standard in quantum theory of atoms in molecules (QTAIM) and is widely used in studies involving topological analysis of electron density [6, 7]. Updated the manuscript for more clarity. (See Page 5,6 and Lines 240 -247)

Please complete conclusions for future experimental work.

We have revised the conclusion section to include recommendations for future experimental studies. (See Page 22, Line 764 – 770)

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Reviewer 3 Report

Comments and Suggestions for Authors

The paper "Targeted Drug Delivery of Anticancer Agents Using C5N2 Substrate: Insights from Density Functional Theory" is an interesting overview related to the C5N2 as carrier for antitumor drugs: cisplatin, carmustine and mechlorethamine by means of DTF analysis.

Abstract: lines 19-21- the interaction energy must be listed.

Introduction: the lines 44-46 must be deleted, there is no relevance for the discussion.

There is a large number of references, somehow some paragraphs must be rewritten.

lines 347-350 - the authors mentioned the highest energy for cisplatin@C5N2, but according to Table 1, it is the lowest!

Figure 3. - the color codes must be mentioned din the legend.

The authors stated that this study will contribute to understanding the delivery mechanism of drugs from C5N2 substrate, but this part is missing in the paper. Even there are discussions related to the interactions of drugs with the substrate, which are the forces and the factors directly involved in the release of drugs?

Based on these observations, I consider that this paper deserves the publication in the Chemistry journal.

Author Response

Reviewer # 3

The paper "Targeted Drug Delivery of Anticancer Agents Using C₅N₂Substrate: Insights from Density Functional Theory" is an interesting overview related to the C₅N₂ as carrier for antitumor drugs: cisplatin, carmustine and mechlorethamine by means of DTF analysis.

Abstract: lines 19-21- the interaction energy must be listed.

As suggested, we have revised the abstract to include the calculated interaction energies for the drug@C₅N₂ complexes.

Introduction: the lines 44-46 must be deleted, there is no relevance for the discussion.

We have carefully reconsidered the content of lines 44–46 and agree that it does not directly contribute to the main focus of the study. Accordingly, we have removed these lines from the revised manuscript to improve clarity and maintain relevance in the introduction.

There is a large number of references, somehow some paragraphs must be rewritten.

We appreciate the reviewer’s valuable suggestion. Following your advice, we have carefully revised several paragraphs to improve clarity and flow while reducing redundancy. We also streamlined the referencing to ensure that only the most relevant and essential citations are included.

lines 347-350 - the authors mentioned the highest energy for cisplatin@C₅N₂, but according to Table 1, it is the lowest!

Upon careful review, we confirm that the interaction energy trend in Table 1 shows that cisplatin@C₅N₂ has the strongest (most negative) interaction energy, indicating the highest adsorption strength, not the lowest. We have revised the wording in the manuscript to clearly state that cisplatin@C₅N₂ exhibits the strongest binding affinity, consistent with the data in Table 1. (See Page 9, Line 375 – 378)

Figure 3. - the color codes must be mentioned din the legend.

We have revised the legend of Figure 3 to clearly explain the color coding: red regions indicate steric hindrance, green regions represent van der Waals interactions, and blue regions correspond to hydrogen bonding. (See Page 11)

The authors stated that this study will contribute to understanding the delivery mechanism of drugs from C₅N₂ substrate, but this part is missing in the paper. Even there are discussions related to the interactions of drugs with the substrate, which are the forces and the factors directly involved in the release of drugs?

We sincerely thank the reviewer for their valuable observation regarding the drug delivery mechanism. In response, we have significantly expanded our discussion in (Section 4, Line 670 – 672) and (Section 5, Line 703 – 709) to explicitly address the forces and factors governing drug release from the C₅N₂substrate. Our new analysis presents:

Quantitative recovery times (cisplatin@C₅N₂: 5.43 × 10⁻⁴ s; carmustine@C₅N₂: 1.22 × 10⁻⁷ s; mechlorethamine@C₅N₂: 1.52 × 10⁻⁸ s at 473 K), revealing distinct release kinetics correlated with interaction energies [1]. Solvent effects, including solvation energies (−65.13 to −46.82 kcal mol⁻¹) and dipole moment shifts, demonstrating how competitive hydrogen bonding in aqueous media accelerates release [2]. pH-dependent behavior, where protonation/deprotonation of C₅N₂’s nitrogen sites modulates electrostatic interactions, particularly for cisplatin (e.g., enhanced release at acidic pH due to weakened coordination bonds).

These additions, supported by references to comparable material systems [3], provide a complete mechanistic understanding of the drug delivery process, fulfilling our original claim about C₅N₂’s tunable release properties.

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