Polylactic Acid and Polycaprolactone Blended Cosmetic Microneedle for Transdermal Hispidin Delivery System
, Jihye Lee 2,†, Kanghyun Choi 2,†, Hyoseon Kim 2, Youngkum Park 2,*, Juhee Yoon 3, June Hyun Kim 4,* and Seongwoo Ryu 1,*Round 1
Reviewer 1 Report
The article is very well written and well organized. It is clear that from each study made maximum information was extracted and the relevant conclusions were drawn. In my opinion the paper entitled "Poly lactic acid and polycaprolactone blended cosmetic microneedle for transdermal hispidin delivery system" might be interesting and useful for researchers in the medical field and I recommend the paper publication in Applied Sciences Journal.
Author Response
We appreciate for your interest in our research works.
Sincerely
Ryu
Author Response File: 
Author Response.docx
Reviewer 2 Report
The study by Lee et al. describes poly (lactic acid) and polycaprolactone blended microneedles (MN) coated with hispidin, for cosmetic application.
Althought nicely designed, the work needs some improvements.
Globally, i) Some English and overall writing should be corrected, ii) The exact applications of hispidin-coated microneedles should be specified, iii) The link between experimental results of the components in solution and hispidin-coated MN should be discussed.
Regarding the results, my main remark concerns Fig. 5 E and F: the results should be quantified and averages from 3 independent experiments shown.
In Figure 6, what is KCTC, and please correct bacteria spelling in A.
For antibacterial activity experiment, why not using hispidin-coated MN directly, instead of hispidin-embedded paper discs?
Below, some details that need correction (the list is not exhaustive):
- Lines 44-46 and 55-57: the sentence is the same, but the references are different
- 90: what is DIW ?
- 96-97: the sentence has to be corrected
- 100: please explain what «fixed» means and how it was done
- 118-119: specify the % of agarose gel and explain how hispidin delivery was measured (instead of putting it in Results section)
- 128: correct «5 × 10+4 cells per well»
- 133: for MTT test, were the crystals dissolved in DMSO ?
- 169: correct the name of bacteria (starts with a capital letter)
- 181: only one concentration of hispidin is mentioned, while more are described in the results section
- 241: to determine the concentration of hispidin at which no cytotoxicity is present, this component was tested in solution. How does it reflect the toxicity of the coated hispidin?
- 298: some strains that are mentioned are not described in Materials and Methods
- 328 : correct the sentence
- 322: biodegradability was not evaluated in the present study
Author Response
Comment 2: The study by Lee et al. describe as poly (lactic acid) and polycaprolactone blended microneedles (MNs) coated with hispidin, for cosmetic application. Althought nicely designed, the work needs some improvements.
Globally, i) Some English and overall writing should be corrected,
As reviewer suggested overall writing is fully revised. Our modification in the article were marked with red colors.
- ii) The exact applications of hispidin-coated microneedles should be specified,
As reviewer suggested the application of hispidin is described in the manuscript.
Our modification:
Therefore, hispidin itself is also a raw material that can be used in cosmetics. It has a superior effect than the existing method of absorbing raw materials through the skin by increasing the skin permeability by using a micro epidermal delivery system. Antibacterial patch, whitening patch, wrinkle improvement patch, etc. can be applied using this.
iii) The link between experimental results of the components in solution and hispidin-coated MN should be discussed.
The transdermal MNs delivery system passes through the epidermis and undergoes biodegradation. Once hispidin is eluted from MNs as solution, the effect of solution blends have similar antibacterial activity. Therefore, we first simulate the antibacterial activity in solution process. We added following description in the manuscript.
Our modification:
The transdermal MNs delivery system passes through the epidermis and undergoes biodegradation. Once hispidin is eluted from MNs as solution, the effect of solution blends has similar antibacterial activity. To determine the concentration of hispidin at which no cytotoxicity was present, PCL, and PLA, B16F10 melanoma cells were treated with hispidin, PCL, and PLA in various concentrations for 24h.
Regarding the results, my main remark concerns Fig. 5 E and F: the results should be quantified and averages from 3 independent experiments shown.
As reviewer suggested, PCR experiments and Western Blot expression experiments were now quantified in the manuscript. However, according to limited samples and experiments, we are unable to represent 3 independent samples. Instead, we added quantification result of figure e and figure f..
Our modification:.
Figure 5. PLA, PCL, and hispidin inflammation in RAW264.7 cells. (a) Cells were treated with different concentrations of PLA, (b) PCL, and (c) hispidin with PLA/PCL, and incubated for 24h. Cell viability was measured by MTT assay. (d) RAW264.7 cells were treated with indicated treatments. Levels of NO production were measured using Griess reagent. (e,f) The RNA and protein expression levels of iNOS and COX-2 were detected by RT-PCR, Western Blot testing and quantification result of each.
In Figure 6, what is KCTC, and please correct bacteria spelling in A.
KCTC stands for Korea Collection for Type Cultures and bacteria spelling in A stands for Staphylococcus epidermidis. We fully revised figure 6 and manuscript for avoiding confusion.
Our modification:
Strains: For the Staphylococcus aureus (Korea Collection for Type Cultures; KCTC 1916), Staphylococcus epidermidis (KCTC 1917), Pseudomonas aeruginosa (KCTC 2153), strain from the Korea Cell Line Bank was used.
Figure 6. Antibacterial Activity determined by Paper Disc Method. (a) Staphylococcus epidermidis, (b) Escherichia coli, (c) Pseudomonas aeruginosa, and (d) Staphylococcus aureus. Hispidin concentrations were 1µM, 5µM, 10µM, and 20µM, respectively. Positive control value was 70% EtOH and negative control was obtained using sterilized DIW.
For antibacterial activity experiment, why not using hispidin-coated MN directly, instead of hispidin-embedded paper discs?
The hispidin-coated MNs could not be directly tested for cytotoxicity in a solid state experiments. Therefore, the original sample was mixed with the experimental group in a liquid state and then the experiment was proceeded. We assume that the biodegradable transdermal delivery system passes through the epidermis and undergoes biodegradation, and then hispidin elutes out of the patch to have antibacterial activity.
Below, some details that need correction (the list is not exhaustive):
We appreciate your comments and reviews. As reviewer suggested, we corrected following marks.
- Lines 44-46 and 55-57: the sentence is the same, but the references are different
As line 44-46 and 55-57 have a redundant meaning, line 55-57 has been deleted.
Our modification:
Recently, Hong Jun Shao et al. demonstrated that hispidin can inhibit the macrophage mediated inflammatory response by down-regulating NF-κB activations [20].
- 90: what is DIW ?
Our modification: deionized water (DIW)
- 96-97: the sentence has to be corrected
Our modification:
PLA (0.08mg) and PCL (0.02mg) were dissolved in DMSO (2.8ml). Then, the temperature was raised up to 100℃ for 24h in a water bath. The solution was double-boiled in a water bath at 100℃ for 24h. After the melting process, the mixed hispidin (2mg) with melted with the PLA/PCL blended polymer.
- 100: please explain what «fixed» means and how it was done
The means of «fixed» are same as dried. So we delited «fixed» word in the sentence. And we added how MNs are fabricated by schematic.
Schematic of this process is added in figure 1a.
Our modification:
The coated MNs were dried for 24h at room temperature.
- 118-119: specify the % of agarose gel and explain how hispidin delivery was measured (instead of putting it in Results section)
Our modification:
After MNs were removed, agarose gel was dissolved into DMSO (111ml). Amount of hispidin in DMSO was measured by UV-Vis and amount of permeability of hispidin was estimated by standard curve comparing with coated amount of pristine hispidin.
- 128: correct «5 × 10+4 cells per well»
Our modification: 5×10⁴cells
- 133: for MTT test, were the crystals dissolved in DMSO ?
Our modification:
The final concentration of DMSO was used at less than 0.1%, which is the concentration that does not affect the cell viability.
- 169: correct the name of bacteria (starts with a capital letter)
Our modification:
For the Staphylococcus aureus (Korea Collection for Type Cultures; KCTC 1916), Staphylococcus epidermidis (KCTC 1917), Pseudomonas aeruginosa (KCTC 2153), strain from the Korea Cell Line Bank was used.
- 181: only one concentration of hispidin is mentioned, while more are described in the results section
Our modification:
The used concentrations of hispidin was 1, 5, 10 and 20μM/disc.
- 241: to determine the concentration of hispidin at which no cytotoxicity is present, this component was tested in solution. How does it reflect the toxicity of the coated hispidin?
The hispidin-coated sample cannot be directly tested for cytotoxicity in a solid state, so the original sample was mixed with the experimental group in a liquid state and then the experiment was proceeded.
- 298: some strains that are mentioned are not described in Materials and Methods
Our modification:
Strains: For the Staphylococcus aureus (Korea Collection for Type Cultures; KCTC 1916), Staphylococcus epidermidis (KCTC 1917), Pseudomonas aeruginosa (KCTC 2153), strain from the Korea Cell Line Bank was used. After adding it to the TSA (Tryptic Soy Agar) medium, the culture was enriched for 18 to 24h at 35 to 37°C. In addition, for Esche-richia coli (KCTC 1039) strain from the same institution was used. After adding it to TSB (Tryptic Soy Broth) medium, the OD value (600nm) was fixed in a range of 0.760 to 0.802 for 18 to 24h at 35 to 37°C in the method previously used (Table 1).
- 328 : correct the sentence
Our modification:
Hispidin with PLA/PCL blends showed a brightening effect of anti-inflammatory activity at the gene levels and high protein levels in skin cell culture experiments.
- 322: biodegradability was not evaluated in the present study
The biodegradability section has been deleted from the text as biodegradability has not been tested.
Author Response File: Author Response.docx
Reviewer 3 Report
(1) Please provide the chemical structure of Hispidin and its physicochemical properties if available.
(2) Please also provide a schematic figure of spin coating on microneedle patch.
(3) What is the rationale of using agarose gel as an in-vitro drug permeation model?
(4) Where is the bacterial clear zone in Fig 6? Could authors zoom in and point them out with arrows in the figure?
(5) Please double-check abbreviations and typos in the manuscript, for example, does DIW mean distilled water?
Author Response
Comment 3:
(1) Please provide the chemical structure of Hispidin and its physicochemical properties if available.
As reviewer suggested we provide the chemical structure of hispidin in the manuscript.
Our modification:
Hispidin (4-O-β-d-glucopyranoside) is a yellowish natural substance present in Phellinus Linteus and is known to be synthesized by a general method. The structural feature is an aromatic compound such as α-tocopherol, which is the same as other phenolic antioxidants, and has a double bond with a benzene ring.
(2) Please also provide a schematic figure of spin coating on microneedle patch.
We added the schematic of spinning coating process in figure 1a.
Our modification:
We fabricated MNs by blending PLA/PCL by direct casting and spinning coating method (figure 1a).
Micro-structure and mechanical strength of MNs. (a) Fabrication process of MNs (b) SEM image of casted pristine PLA/PCL MNs. (c) Stress-strain graph of various PLA/PCL blended structures.(d) SEM image of MNs after coating of hispidin delivering layer. (e) SEM image of MNs after coating of hispidin with PLA/PCL blended polymer delivering layer.
(3) What is the rationale of using agarose gel as an in-vitro drug permeation model?
According to previous research works, agarose gel is a homogeneous and semi-clear substance which is mimicked porcine skin. Therefore, agarose gel could be used as a skin transitional substance that has the same viscoelastic properties (J Pharm Sci, 2014, 103(2) 613-27). We modified previous models as a target for an estimate the amount of hispidin and PLA/PCL from MNs. We added following previous model as a reference for our experiments.
Our modification:
Agarose gel was used to measure the permeability of hispidin into the skin. Agarose gel is a homogeneous and semi-clear substance which is mimicked porcine skin for skin transitional substance [21]. The coated MNs were applied to the agarose gel (3g) for 5min with hand pressure.
- hang D; Das DB; Rielly CD. Microneedle assisted micro-particle delivery from gene guns: experiments using skin-mimicking agarose gel. J Pharm Sci. 2014 103(2) 613-27.
(4) Where is the bacterial clear zone in Fig 6? Could authors zoom in and point them out with arrows in the figure?
Bacterial clear zone is pointed out with arrows in figure 6.
(5) Please double-check abbreviations and typos in the manuscript, for example, does DIW mean distilled water?
As pointed out by the reviewer, abbreviated expressions were revised in the text.
Our modification: deionized water (DIW)
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
All the questions/remarks were addressed.
