Levofloxacin and Amikacin Adsorption on Nanodiamonds: Mechanism and Application Prospects

why choose the two broad-spectrum antibiotics of amikacin and levofloxacin in this study?

Amikacin and levofloxacin were chosen for such study because of several reasons.

1. We tried to reveal the influence of the sorbate charge on its ability to be adsorbed on nanodiamonds. Amikacin was used in the form of sulfate; therefore, it acts as a cation and levofloxacin is an acid.

2. Nanodiamonds are considered as universal drug carrier. The antibiotic should be chosen by the physicians for the patient tailored to individual patients and circumstances antibiotic prescribing. Therefore, we need to compare different drugs in the relationship to nanodiamonds.

Moreover, we both drugs are broad-spectrum antibiotics with known pharmacodynamic parameters.

What are the current limitations of antibiotics in cardiovascular surgery?

In most cases antibiotics that are used in cardiovascular surgery will take a few hours to work, while recent systematic reviews and meta-analysis of randomized controlled trials have concluded that surgical site infection can be reduced by prolonging prophylaxis for 24–48 h. Also, post-operative pneumonia and all-cause mortality can be reduced by giving agents with both anti-Gram-negative and anti-Gram-positive activity. The choice of the most appropriate regimen remains open to debate. [J Antimicrob Chemother 2012; 67: 521–522]. Loading of carriers with antibiotics is expected will provide prolonged release of the drug.

Among the antimicrobial effects, only DND coating, DND-Amikacin coating and DND-Levofloxacin coating were shown, but the antibacterial effects of Amikacin and Levofloxacin alone were not shown

Both amikacin and levofloxacin are widely used as antimicrobial agents. For free levofloxacin as well as amikacin minimum inhibitory concentration required to inhibit 90% of Streptococcus is close to 1 mg/L [Expert Rev. Anti Infect. Ther. (2010) 8(11), 1259–1271; J. Infectious Diseases. (1976) 134, S297-S307; The Immunoassay Handbook (Fourth Edition) 2013, 5-962]. Therefore, in the present study we need to confirm does antimicrobial activity persist after adsorption on nanodiamonds.

This paper is limited to the antimicrobial experiments of Staphylococcus aureus in vitro, and no experiments have shown the antimicrobial effects in vivo. But the title of the paper says "Prospects of application in cardiovascular Surgery"

In vivo experiments were performed for nanodiamond coatings, and nanodiamond-drug composites are under the long-term animal test right now. Since we have shown the possibility of formation of stable adsorption complex and preservation of antimicrobial activity after the adsorption, we entitle our study as “Prospects”.

Although the dose of antibiotics can be reduced, what is the safety of DND?

There are a large number of experimental studies and reviews that demonstrate low toxicity of nanodiamonds and even suggest nanodiamonds as carriers in the development of drug delivery systems. Therefore, we believe that nanodiamond as a base of biotissue coating is a perspective tool of nanodiamond medical application.

Recent technical advancement for drug adsorption on nanodiamonds has been large advanced. The references details are as follows:

Mochalin, V. N., et al. "Adsorption of drugs on nanodiamond: toward development of a drug delivery platform. " Mol Pharm 10.10(2013):3728-3735.

Wang, L., et al. "Safety evaluation of nanodiamond-doxorubicin complexes in a Nave Beagle canine model using hematologic, histological, and urine analysis." Nano Research (2021)

Salaam, A. D. , et al. "Nanodiamonds enhance therapeutic efficacy of doxorubicin in treating metastatic hormone-refractory prostate cancer. " Nanotechnology 25.42(2014):425103.

Wang, Xin , et al. "Epirubicin-Adsorbed Nanodiamonds Kill Chemoresistant Hepatic Cancer Stem Cells." Acs Nano 8.12(2014):12151-12166

All references are included in the revised version of the manuscript

After all, the safety of nanodiamonds is very important. Authors are encouraged to discuss the strengths and weaknesses of nanodiamonds in the results section, as well as objectively evaluate the strengths and weaknesses of drug adsorption on nanodiamonds.

Please, see the revised version of the manuscript. The main limitations and weaknesses of nanodiamonds medical application can be reduced to the following:

If diamond nanoparticles get in the bloodstream, it gets accumulated majorly in lung, spleen and liver and finally gets excreted into the urinary tract. Such behavior might result in an inflammatory response in the lungs and high dose- dependent retention of NDs in the lung [J. Phys. Chem. B 111 (2007) 2-7]. On the other hand, if nanodiamond is used in form of hybrid composite e.g. nanodiamond-lipid it shows some histological alterations and changes in blood biochemical parameters affecting the liver function and lipid metabolism, but no organ dysfunction and signs of cell destruction [Adv. Mater. 2013, 25, 3532–3541].

The process of complexing nanodiamond with active moiety through covalent bonding is a complicated process and it is difficult to eliminate the toxic solvents used during synthesis. Moreover, the complex formed cannot show slow-release function [J. Pharmaceutical Analysis. 10 (2020) 1-12]. These problems can be solved by application of adsorption complexes of nanodiamonds. Since adsorption process is usually performed from an aqueous phase, it excludes the adsorption the toxic solvents. Coulomb attraction is usually the main driving force of the adsorption and complex formation. Ionic and hydrogen bonds are controlled by ionic strength and pH that provide the possibility of control drug release.

Why the DND is in an aggregate state containing primary particles with a diameter of about 4-6 nm?

Detonation of oxygen-deficient explosives in an inert medium produces ultra-fine diamond articles with a diameter of about 4–6 nm. The reason for the difficulty is that the core aggregates having a diameter range of 100–200 nm are extremely tight and could not be broken up by known method of de-aggregation [Carbon 43 (2005) 1722–1730]. Therefore, we characterize the suspension that we have purchased from PlasmaChem by means of different analytical methods that are described in the manuscript.

Figure 1 caption is too simplized. The authors should add more description about the number and intensity values

The caption was modified in accordance with Reviewer recommendation.

Did the authors check the pH effect on the adsorption of the drug loading and release?

In the present study we have not checked the influence of pH, but ionic strength and the presence of the desorbing agent was analyzed.

The detailed functional groups on the surfaces of DND are not clear

Therefore, the surface of DND used in our study coated with carboxyl groups that provide negative surface charge, and other oxygen-containing groups including hydroxyl, ester and anhydride.

The toxicity of DND is not checked for biomedical applications

If diamond nanoparticles get in the bloodstream, it gets accumulated majorly in lung, spleen and liver and finally gets excreted into the urinary tract. Such behavior might result in an inflammatory response in the lungs and high dose- dependent retention of NDs in the lung [J. Phys. Chem. B 111 (2007) 2-7]. On the other hand, if nanodiamond is used in form of hybrid composite e.g. nanodiamond-lipid it shows some histological alterations and changes in blood biochemical parameters affecting the liver function and lipid metabolism, but no organ dysfunction and signs of cell destruction [Adv. Mater. 2013, 25, 3532–3541].

The process of complexing nanodiamond with active moiety through covalent bonding is a complicated process and it is difficult to eliminate the toxic solvents used during synthesis. Moreover, the complex formed cannot show slow-release function [J. Pharmaceutical Analysis. 10 (2020) 1-12]. These problems can be solved by application of adsorption complexes of nanodiamonds. Since adsorption process is usually performed from an aqueous phase, it excludes the adsorption the toxic solvents. Coulomb attraction is usually the main driving force of the adsorption and complex formation. Ionic and hydrogen bonds are controlled by ionic strength and pH that provide the possibility of control drug release.

This part was added in the revised version.

The authors should present the stability of DND in the presence of albumin solution (whether there is size change with/without albumin).

Addition of saline results in the aggregation stability lose, while the addition of albumin in saline reduces the size distribution to the initial values. The corresponding figures are added into the revised version of the manuscript.

The title of the manuscript is misleading. There’s no evidence shown in the manuscript that the materials can be used for cardiovascular surgery. In vitro (cell-based assays) or in vivo (animal models) of cardiovascular disease should be used to prove that the material is compatible with the cardiovascular surgery process. Otherwise, the title should be revised.

The title is revised in the revised version

In Figure 2, a lower concentration of DAD should be used to show individual nanoparticles clearly. Also, the aggregation of DAD in solution should be discussed to explain the discrepancy between TEM and DLS

Figure is changed in the revised version. Detonation of oxygen-deficient explosives in an inert medium produces ultra-fine diamond articles with a diameter of about 4–6 nm. The reason for the difficulty is that the core aggregates having a diameter range of 100–200 nm are extremely tight and could not be broken up by known method of de-aggregation [Carbon 43 (2005) 1722–1730]. Therefore, we characterize the suspension that we have purchased from PlasmaChem by means of different analytical methods that are described in the manuscript. For DLS measuring performed the suspension was diluted to final concentration of 1 mg/ml with water, saline and 40-g/l serum albumin solution in saline. Saline and serum albumin solution were used as a desorbing media for drugs. Therefore, it was important to reveal its influence on DND particles size distribution. It was observed that addition of saline results in the aggregation stability lose, while the addition of albumin in saline reduces the size distribution to the initial values. The average particle size in the suspension is close to 100 nm for the original DND.

From Line 299 to Line 307, desorption experiment should be revised to provide more information of the process. For example, desorption kinetics should be measured in water and saline. Also, stability of DAD and drug-loaded DAD in the solution should be tested by measuring change of size over time

Our previous results with amikacin included long-term desorption [Mend. Comm. 29 (2019) 318-319] indicates that 48 h is sufficient time for the onset of equilibrium. On the other hand, 48 h is twice as long as the time to determine the survival of bacteria. So, this period was chosen for desorption analysis. Since nanodiamond-drug composite are considered as material for preparation of coverage of the collagen tissue, changes in the particles size over time in the aqueous suspension during the desorption process unlikely to be an important parameter.

In Figure 5 and Table 3, a statistical analysis should be performed to show the results are statistically significant.

In the revised version the data are presented with a confidence interval.

In Figure 5 and Table 3, different doses of the materials should be tested to show whether the response is dose-dependent

The goal of the present was to show does amikacin and levofloxacin preserve its antimicrobial activity being adsorbed on nanodiamonds. Our previous study shows that drug can lose its activity after adsorption on nanodiamonds [Mend. Comm. 27 (2017) 421-423]. Since antimicrobial activity is preserved such tests will be provided as well as an animal trials.

In terms of antimicrobial characterization, free antibiotics should be used as a control group

Biocompatibility is a major concern of nanomaterials. The authors should test the biocompatibility of the as-prepared materials using cell lines or animals

Both amikacin and levofloxacin are widely used as antimicrobial agents. For free levofloxacin as well as amikacin minimum inhibitory concentration required to inhibit 90% of Streptococcus is close to 1 mg/L [Expert Rev. Anti Infect. Ther. (2010) 8(11), 1259–1271; J. Infectious Diseases. (1976) 134, S297-S307; The Immunoassay Handbook (Fourth Edition) 2013, 5-962]. Therefore, in the present study we need to confirm does antimicrobial activity persist after adsorption on nanodiamonds.