如题，投了这个期刊两个半月审稿给了大修，但感觉很多内容不知道咋改大神帮忙看看修改后接受机率大不大，第一个审稿人一堆意见，第二个几乎没有啥太多问题= =
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Reviewers’ Comments to Author:

Reviewer: 1

Comments to the Author
Article's topic is interesting
As far as I am concerned, it should have been submitted as a short communication since the microsphere's characterization is not optimal. However these vectors have been deeply characterized since the  middle of the 90's. By taking into account the characterization, several other experiments could have been carried out in order to characterize, understand and explain under a deeper way, the active ingredient releases and  mechanism. Characterization such DSC, TGA, porosity should have been done to correlate the dissolution mechanism and the microspheres physico-chemical behaviour.
Introduction:references are adequate although more recent ones are available. Several other techniques exist for microsphere preparation , such as spray-drying, that represents a single step, scaleable method, widely used in industry. The fact that pesticide microspheres has been manufactured and is not well known on the literature should have been more underlined and highlighted.
Same things for PBS polymer that is not as known as PLA, emphasis should have been put on its biocompatibility and biodegradability, especially since these particles are intended to be used in the environment.   
There is no mentioned of polyvinyl alcohol carcinogenicity in the introduction whereas several patents exist in that regards.

Experimental

Materials: PLA and PBS are not characterized at all: no Mw, no Mn, no polydispersity. Same thing for PVA : is it hydrolyzed, partially hydrolyzed,......what is the Mw?
Why a 35 000 spec has been selected? Sink conditions?,....

Preparation of microspheres
When  microspheres were washed 3 times, this  may be one of the explanation why the loading efficiency s not 100%, water extracting the active ingredients that are adsorbed/at the surface/into the pores that are close to the surface.
Azoxystrobin and difenoconazole assays should have been done in order to determine the impact of emulsion, and water washing on microsphere's loading efficiency.   
Manufacturing preparation should also have been tested by saturating water phase with  azoxystrobin and difenoconazole to see how far it may have impacted positively the loading efficiency.
Dosing of dichloromethane (DCM) , a class 2 solvent (www.ich.org), with a PDE of 6 mg per day and 600 ppm max threshold, should have been carried out by GC. It is already known and published that DCM is pretty difficult to remove, even by freeze drying. Desiccator does not vacuum enough. Furthermore, ethyl acetate and other class 3 solvents have already been used to prepare microspheres. It would have been a good idea to demonstrate the feasibility of using this class 3 solvent without changing the release mechanism and particle physico-chemical characterization. It is already known that ethyl acetate will generate pores and higher pore families, being more hydrosoluble than DCM....fine tuning should then be expected to get the desire mechanism.   
The ratio PBS and PLA is of great interest however Tg and thermal properties of the polymer alloy have not been assessed and would have been useful to predict the stability and the physical behaviour of the microsphere (whether there was a plasticizing effect, generating higher porosity....)

Particle size distribution
It would have been interesting to monitor the impact of ultrasonication on particle size and particle size distribution. A sentence in that regards would have been relevant.

Drug loading and encapsulation efficiency
Section is OK however as mentioned earlier, the impact of washing should have been monitored (assay before and after washing).

Acute toxicity on zebra fish.
Zebra fish is known to be a relevant model to determine toxicity.  Animals should have been treated in accordance of any council for animal care. Even though LD 50 has been assessed based on the guidelines, a maximum tolerated dose could have been monitored also, LD50 and CL not being used anymore since 1992.   
Standard of empty microspheres, free pesticides should have been part of the toxicity test .

In vitro release studies
It is not explained why a mixture of 50:50 methanol/water mixture has been used. Furthermore the hydrodynamical conditions has not been explained (rpm? 25C?). What is an appropriate interval? Based on what? The fresh methyl alcohol buffer is the 50-50 mixture? It is not a buffer or please show the buffering capacity. Dissolution results would have been different if water, or phosphate/carbonate buffer have been used. Samples should have withdrawn in triplicate. At then end of dissolution test, particle should have been dried and tested for active substance assay in order to reconciliate the release and the drug loading. Last question: in the Figure 6, 100% was not reached because percentages have been expressed in drug loading efficiency, being less than 100%  so it is not possible to reach 100% ?

Results and discussion
Discussion with regards to the smooth surface could have been more detailed. There is no procedure for the EM analysis (type, gold coating unit,....) . Furthermore, the nice obtained microphotographs showing smooth surface may be interpreted as an artifact since it is difficult to determine whether the gold coating may have filled the pores localized at the surface of the microspheres.

Morphology and particle size distribution of the microspheres
The sentence with the pores could have been more detailed. A lot of publications exist in that regards and this the reason why it has been proposed earlier to carry out some porosity experiments.

IR spectrum
There is no details with regards to potential interactions between active substances and polymer alone or alloy. This may  explain why the release percentages did not reach 100%? or it is because the loading efficiency was slower than 100%? A covalent  interaction may not be measured by FT-IR .... NMR or solid state NMR would have been useful.

Toxicity evaluation
Particle size and particle size distribution would have been of interest to assess toxicity correctly.  Microspheres showing different particle size  should have been used to monitor this last assumption. As mentioned earlier, empty microspheres and free active substances should have been tested as positive/negative standard. Microspheres showing different release should have been tested to determine more accurately the impact of drug release on toxicity. There is no correlation in the discussion between free and encapsulated active substances : when could we expect tox? what is the max release rate?,....
Furthermore these two active substances are hydrophobic since dissolved in the organic phase therefore the release rates may be solubility dependant, meaning that hydrophilic substances or pesticides may show faster dissolution rate. Thermal analysis could have explained whether the pesticides were molecularly or particularly dispersed. Particularly dispersed hydrophobic substances would have shown the same dissolution rates than hydrophilic substances, substances being molecularly dispersed therefore being already in solution. Plasticizing effect may have been monitored by DSC by decreasing the Tg that  may have explained more deeper the release mechanism vs temperature.   

Drug loading , encapsulation, efficiency and drug release study
It is not mentioned whether the sink conditions were reached. The high burst effect has already been published and explained, and were not correlated with toxicity. Not enough different formulations have been tested to show that it was possible to control the release rate and to control microsphere's characteristics.

Conclusion:
More detailed conclusion with regards to what should be done next  was expected. Based on the above comments, the conclusion could be built in that regards.   


Reviewer: 2

Comments to the Author
Manuscript concerns the preparation and release properties as well as toxicity of PBL/PLA microspheres containing fungicide mixture (azoxystrobin and difenoconazole). Obtained microspheres characterized prolonged release of fungicides and relative low toxicity in zebrafish test. Paper is interesting and should be publish after several improvements:
1.Reading the description of the 2.4 chapter I was slightly confused. Authors at first … “dissolved specific amount of dried samples (microspheres???) in methylene chloride to confirm liberation of drug molecules from PBS and PLA polymers and then left some time for complete evaporation. After that, these samples were dissolved in methyl alcohol. The solution was then collected and analyzed using …. HPLC”.
The question arises whether pesticides used completely do not mix with polymers?? If they mix (even partially), what really dissolved in methanol? After all PLA/PBS does not dissolve in methanol and methanol will not extract the pesticide from the blend. The presented procedure should be explained in the revised version.
2. Molar mass of the polyester used should be added. I think that supplier attached such data as Mn and Mw/Mn. Moreover it should be also clearly said which PLA crystalline – PLLA or amorphous – PDLA) was used for experiments.
3. I understand that due to low solubility of fungicides in water (several mg/L) methanol was used in drug release study, however, in studied system (biodegradable polymer) mixture methanol/water should be better solvent because polyester degradation affects the drug release rate. I understand that release measurements presented in the manuscript show prolonged drug release what was the aim of the authors but effect of water when aliphatic polyesters are used will be notable.
4. Information concerning PLA/PBS miscibility should be mentioned in Introduction or comment in discussion. 返回小木虫查看更多