Branched amphotericin functional poly(N-isopropyl acrylamide): an antifungal polymer

Branched poly(N-isopropylacrylamide) was functionalized with Amphotericin B (AmB) at the chain ends to produce an antifungal material. The polymer showed antifungal properties against AmB-sensitive strains of Candida albicans, Fusarium keratoplasticum and Aspergillus flavus (minimal inhibitory concentration ranged from 5 to 500 µg ml−1) but was not effective against an AmB resistant strain of C. albicans nor against Candida tropicalis. The polymer end groups bound to the AmB target, ergosterol, and the fluorescence spectrum of a dye used as a solvatochromic probe, Nile red, was blue shifted indicating that segments of the polymer became desolvated on binding. The polymer was less toxic to corneal and renal epithelial cells and explanted corneal tissue than the free drug. Also, the polymer did not induce reactive oxygen species release from peripheral blood mononuclear cells, nor did it cause a substantial release of the proinflammatory cytokines, tumour necrosis factor-α and interleukin-1β (at 0.5 mg ml−1).

ergosterol binding to PNIPAM-AmB induces a phase change of the whole material (i.e. the coilglobule transition), when I understand there is really some dehydration of AmB end groups. I suggest this statement is revised.

Review form: Reviewer 2 (Nick Turner)
Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
This is a really interesting paper and i enjoyed reading it. I am recommending publication with minor cosmetic revisions. P2, L3 "less toxic" than what"? Recommend this single sentence be more specific. Figure 1: The use of a,b,c in superscript was difficult to see and could be confusing to nonspecialists (who may not understand MW a is actually MW with an annotation. Recommend using *,$,# instead.   Figure 3: legend. P<0.001 is missing its *** Figure 4:Check colours of * vs those on figures. Consider rewording the significance sentence as it is a little confusing (suggest removing colours and just use *,**,*** in black and description) . See Figure 5 for clear example. I think reader will realise that the * are colour coded to lines in the figures. Same for other figures (6+7)> Decision letter (RSOS-201655.R0) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Miss Rimmer:
Title: Branched Amphotericin Functional Poly(N-isopropyl acrylamide): an Antifungal Polymer Manuscript ID: RSOS-201655 Thank you for submitting the above manuscript to Royal Society Open Science. On behalf of the Editors and the Royal Society of Chemistry, I am pleased to inform you that your manuscript will be accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the reviewers' comments at the end of this email.
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Comments to the Author: (There are no comments.) ********************************************** Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) This paper by Prof Rimmer and co-workers describes the utilisation of their branched PNIPAM materials as antifungals. The work is novel, the results are scientifically interesting and the findings medically important. I believe this paper will be of interest to the readers of Open Science. I do have some suggestions for revision prior to publication.
Page 3. Line 9-11-"mortality rates as high as 60% with Candida sp. infections." This could be rephrased to make clear that this is (presumably) a mortality rate for certain infections, rather than an estimate for the average infection of any candida type. Page 3. Line 34 onward: The authors move to a personal tense (e.g. "We") -the editor can recommend if this should be moved to an impersonal tense. Page 5, Line 49: The authors state that the coil-globule transition occurs at the LCST. At this point the use of LCST and Tc-g seems to vary in the manuscript. E.g. "…increase in Tc-g; from a peak of 18.9 °C for the as prepared pyrrole-functional material" compared to "LCST measurements were carried out on a Nano DSC by TA Instruments" (SI, 3.). LCST used in the methods section in the main paper. If these are used interchangeably, I think a single designation would be clearer. Page 7, Section "HB-PNIPAM-AmB is active against fungi" -it would be useful to clarify if the MIC is calculated based on the moles of AmB in the polymer, or the moles of polymer itself. I assumed moles of AmB in the polymer-AmB solution, but then the cytotoxicity experiments, which I assumed were conducted based on polymer concentration, asked for comparison to the MICs ("even at 5 mg mL-1, which is far in excess of the MIC data"). I think this confusion arises because the weight-based MIC appears to be related to polymer concentration, whereas the molar MIC is based on conc of AmB in the polymer. I guess this is the case because the only way I could get the numbers to make sense is convert weight to moles using this conversion factor given in the table in figure 1. I recommend making this conversion clear in the table headings and caption. Page 7. "AmB compared with the cell only control (…. 100 ± 5.2 %)" It is not very clear to me from the methods how you determined error on these measurements of control if the data are normalised to these healthy cells. Page 8, figure 3. The caption is written in a confusing manner. "Cell viability, relative to untreated controls, either in the absence of polymer or in the presence of AmB:" I guess these comments relate to the controls, but it is written in a confusing manner. Then for A,B and D,E the two test samples are separated, but placed together in C. I would suggest revising the figure caption and splitting element C to make this easier to follow for the reader. Page 17. "HB-PNIPAM-AmB both bound to ergosterol and was effective against a range of bacteria" -I assume this is meant to read "fungi" Page 18. "Synthesis of HB-PNIPAM-AmB": "HB-PNIPAM with COOH end groups prepared as previously described.36" -I understand that this synthesis is described in the SI under "Synthesis of HB-PNIPAM Precursor." It would be easier to follow with a reference to the SI and a consistent terminology used for this polymer. This method could also include the LCST of the final product, for consistency. Page 19 "Overnight cultures were adjusted to an optical density at 600 nm of 0.1 and incubated with Amphotericin polymer, which was serially diluted 1:2 from 2500-2.44 µg mL-1, for 16 hours in a 96 well plat" -it would be useful to state the incubation temperature (I guess 37C), especially given that the LCST seems to be very close to 37C. I would imagine the LCST transition may affect MIC. Page 20. "Rabbit limbal epithelial cell viability" and "Human Renal epithelial cell viability"whether data are compared using one or two way ANOVA should be stated. Page 21, Conclusions. "In conclusion, highly-branched poly(N-isopropyl acrylamide) with AmB end groups binds to ergosterol with a desolvation phase transition". This implies to me that the ergosterol binding to PNIPAM-AmB induces a phase change of the whole material (i.e. the coilglobule transition), when I understand there is really some dehydration of AmB end groups. I suggest this statement is revised.

Reviewer: 2
Comments to the Author(s) This is a really interesting paper and i enjoyed reading it. I am recommending publication with minor cosmetic revisions. P2, L3 "less toxic" than what"? Recommend this single sentence be more specific. Figure 1: The use of a,b,c in superscript was difficult to see and could be confusing to nonspecialists (who may not understand MW a is actually MW with an annotation. Recommend using *,$,# instead.    Comments to the Author(s) This paper by Prof Rimmer and co-workers describes the utilisation of their branched PNIPAM materials as antifungals. The work is novel, the results are scientifically interesting and the findings medically important. I believe this paper will be of interest to the readers of Open Science. I do have some suggestions for revision prior to publication.
1. Page 3. Line 9-11-"mortality rates as high as 60% with Candida sp. infections." This could be rephrased to make clear that this is (presumably) a mortality rate for certain infections, rather than an estimate for the average infection of any candida type.

Answer: In one of the studies we cited there is no mention of the types of underlying infections and the study refers to only nosocomial infections.
We have added this and we now cite the primary study as well the review paper. We added some other primary studies, which refer to general issues with patients needing critical care. It does seem to us that we could point to patients with HIV cancer or cardiovascular underlying conditions or probably SARS and complications from fungal colonization. However, It seems more useful to follow the clinical studies, which mostly are concerned with the generality of the susceptibility of critically ill patients. We rewrote the text as requested as follows: " However, fungal infections are of great importance due to complications in other diseases. 5 For example recent studies (in Canadian ICUs) showed upto 50% of critically ill patients were colonised with Candida sp. 6, 7 and mortality rates as high as 60% in nocosomial infections have been reported. 6, 8, 9 " 2. Page 3. Line 34 onward: The authors move to a personal tense (e.g. "We")the editor can recommend if this should be moved to an impersonal tense.
P2, L3 "less toxic" than what"? Recommend this single sentence be more specific.
Answer: The sentence is : " A branched polymer with amphotericin end groups is active against fungi but is less toxic than Amphotericin-B." so this indicates the polymer is less toxic than Amphotericin-B. Figure 1: The use of a,b,c in superscript was difficult to see and could be confusing to non-specialists (who may not understand MW a is actually MW with an annotation. Recommend using *,$,# instead. Answer: this is a good point and we have changed the supersripts to be less confusing.  Answer: this has been changed as requested Figure 3: legend. P<0.001 is missing its *** Answer: this has been added as requested Figure 4:Check colours of * vs those on figures. Consider rewording the significance sentence as it is a little confusing (suggest removing colours and just use *,**,*** in black and description) . See Figure 5 for clear example. I think reader will realise that the * are colour coded to lines in the figures.