Conjugation of Urokinase to Water-Soluble Magnetic Nanoparticles for Enhanced Thrombolysis

Round 1

Reviewer 1 Report

This manuscript describes ferric oxide conjugated urokinease nanoparticles for targeted delivery as thrombolytic.  The idea is good but ferric oxide is not biodegradable which brings up a major concern.  A few specific comments are as below.

Ferric oxide nanoparticles will not be soluble as mentioned in the abstract. 50 nM particles will not be cleared by kidney. Particle charge will be responsible for aggregation or de-aggregation too. Fig 3 has no information on replicates, no SD bar Only BCA assay will not give the information on the stability of urokinase Why gastric epithelial cells – the product is supposed to be used IV There is some concentration dependent cell death in fig 5 – why ? Fig 2 (f) does not show much difference of urokinase release with or without magnetic field

Author Response

Thank you for your valuable suggestions and comments. Below are the point-by-point responses to your comments.

Thank you for your kind reminder. 50 nm particles will not be directly cleared by kidney because their size exceeds the glomerular cut-off size (about 6 nm). Recently, Naumenko et al reported that large non-filtered NPs can be translocated through endothelial cells and epithelial cells in the renal tubules and get excreted viaurine (Intravital microscopy reveals a novel mechanism of nanoparticles excretion in kidney, Journal of Control Release, 2019, 307:368-378). They found that particles with 140 nm can be transported to the outside. If so, the particles with mean size of 50 nm can also be cleared in the same way. However, the related content will not be discussed in this paper. The SD bar has been added to Figure 3 in the revised version. The stability of urokinase is discussed in Section 3.1. As shown in Figure 7, in the absence of AC magnetic field, the release of urokinase is only about 56 μg after 60 min. There are 92.6% of the initial amount of urokinase still binds to NPs. This indicates that the prepared urokinase coated magnetic NPs are very stable. We are sorry that the gastric epithelial cells were used in the toxicity assay. In the revised version, human endothelial cells (C6246, Beyotime, Shanghai, China) were adopted for the toxicity test. The assay results of cell activity under the action of urokinase@PMAO-OA-Fe₃O₄ NPs within 72 hours are shown in in Figure 5. We are not quite sure about the reason of the concentration-dependent cell death. According to the research results of Naqvi et al (Concentration-dependent toxicity of iron oxide nanoparticles mediated by increased oxidative stress, 2010, 5:983), the oxidative stress related to the concentration of iron oxide nanoparticles can induce cell injury and death. Although the surface coating increases the safety of iron oxide nanoparticles in our method, oxidative stress may still affect the viability of cells. Figure 2f is for explaining that the magnetic separation characteristic of the nanoparticle solution can be confirmed by detecting its transmittance after being subjected to an external magnetic field for a certain period of time. The magnetic field used here is static. Static magnetic field will not have much effect on the release of urokinase. Therefore, whether there is a magnetic field or not, there is not much difference in the release of urokinase.

Reviewer 2 Report

The paper "Conjugation of Urokinase to Water-soluble Magnetic Nanoparticles for Enhanced Thrombolysis " presents an experimental study of  water-soluble magnetic nanoparticles. The experimental investigation and analysis are well done and scientifically sound but some parts of paper need correction. Thus the paper is suitable for publication in Applied Sciences after the following points have been carefully addressed (minor revisions and comments):

First of all, I advise to separate experimental part from results. Fig 2e – FTIR resuts. Peaks at around 2800 cm-1 should be described. “As shown in Figure 2f, the transmittance of these solutions increases from 0% to more than 80% from 0 to 100 min” At with wavelength optical transmittance was measured?  Fig 2f. Caption suggests magnetic properties while optical transmittance was measured. Fig. 5 and 9. How uncertainty was calculated? XRD results. Provide literature date (card numbers) from with peaks was decried (Fig. 2d)

Author Response

 

 

Author Response File: Author Response.doc