Development of Folic Acid-Conjugated and Methylene Blue-Adsorbed Au@TNA Nanoparticles for Enhanced Photodynamic Therapy of Bladder Cancer Cells
Round 1
Reviewer 1 Report
Summary
This paper presents the synthesis of gold nanoparticles by reduction of Au(III) by tannic acid. Then methylene blue (MB) was adsorbed and folic acid was then grafted onto the surface of nanoparticles using a PEG3500. These Np were characterized with TEM which showed the formation of a mixture of both spherical and triangular nanoparticles.
A spectroscopic study was carried out as well as experiments with living cells to demonstrate a PDT activity.
Review:
In the paper, there is no bibliography about tannic acid gold nanoparticle synthesis. It would be interesting to discuss about the benefit of room temperature condition, as in the literature there are several examples of gold nanoparticle synthesis with tannic acid but usually with additives (reducing agents) and heating. Tweaking these parameters might have been useful to produce more homogenous nanoparticles.
P3. Preparation of Au@MB NPs
There is no reaction time indicated.
Authors should indicate the deionized water as the solvent (even if it seems obvious). MB mother solution was in water too?
L119 seems to suggest that the addition lasts 24h, I guess that this is not the addition time but, but the reaction time.
It would have been interesting to provide the absorption spectrum of the last centrifugation supernatant after MB adsorption.
P5 202: The 528 and 546 nm band are not interband transitions. Interbands in gold don’t appear as a peak but as large absorption band below 450nm. The maximum absorption at this wavelength arises from localized surface plasmon resonance LSPR.
P5 204 “refexion index” à refractive index
P5 L210 : It’s difficult to discriminate well the absorption peak of MB because of the prominent extinction of gold nanoparticles. Moreover, absorption peak of MB is very variable depending on solvent polarity and hydrogen bond it can vary in presence of tannic acid and PEG.
P5 L212-218: The presence of MB fluorescence during RAMAN experiment may also be induced by released free MB in solution. It’s very difficult (nearly impossible) to be sure that no MB is free in solution even after several purification cycles with centrifugation. Since fluorescence is very sensitive and MB is quite fluorescent very few free MB can lead to this result. Moreover, even well purified nanoparticles can slowly release MB.
P6 Figure 2b in the caption there are two captions for figure 2b (fluorescence spectra or SERS measurement).
P6 Singlet oxygen generation assay. I don’t understand why using RNO/imidazole, such a complex 1O2 probing system implies two successive reactions which can lead to errors. (https://doi.org/10.1007/s00216-019-01910-2). Moreover, RNO is a very reactive specie.
The singlet oxygen quantum yield measurement method is wrong. There is no equation presented to explain the calculation of this quantum yield, although it is a quite complicated measurement. Methods for singlet oxygen measurement are described in other papers which use DPBF or water soluble anthracene derivatives. Some experimental considerations have to be taken. The consumption of the quencher has to be negligible to ensure that there is no change of quencher concentration during all experiment (indeed when the quencher concentration is lowered, its ability to react with singlet oxygen is lowered since singlet oxygen deactivate itself in the solution before reaching the quencher). Due to method errors, the singlet oxygen generation quantum yield by Au@TNA@MB is very over evaluated by comparison with MB alone.
P6 L237 : Thermographic image are depicted in figure 3c and not a figure with MB photostability.
P7 figure 4 c) it gives the impression that cells were treated during 4h under irradiation.
Author Response
Reviewer 1
- Preparation of Au@MB NPs
There is no reaction time indicated.
Response: We thank for reviewer’s comment. The reaction time has been added in the Experiment Section. Please see page 3, line 122.
- Authors should indicate the deionized water as the solvent (even if it seems obvious). MB mother solution was in water too?
Response: We thank for reviewer’s comment. DI water was used as the solvent to dissolve all hydrophilic chemicals. The relevant description has also been added in the main text. Please see page 3, lines 118.
- L119 seems to suggest that the addition lasts 24h, I guess that this is not the addition time but, but the reaction time.
Response: We thank for reviewer’s comment. To make the description clear, we modified the sentence to present the reaction parameter in the corresponding experiment section. Please see page 3, lines 126 and 127.
- It would have been interesting to provide the absorption spectrum of the last centrifugation supernatant after MB adsorption.
Response: We thank for reviewer’s comment. We measured the 1st and last supernatant solutions from mixture of MB and Au@TNA nanoparticles after 24 h of mixture time collected from centrifugation. The experiment method, relevant sentence, and data result have been added in the revised manuscript. Please see Figure S2, page 3, lines 129-131, and page 4, lines 138-141, and page 6, lines 221-222.
- P5 202: The 528 and 546 nm band are not interband transitions. Interbands in gold don’t appear as a peak but as large absorption band below 450nm. The maximum absorption at this wavelength arises from localized surface plasmon resonance LSPR.
Response: We are sorry for the scientific mistake. We have revised the interband transition as '' localized surface plasmon resonance LSPR ''. Please see page 6, line 218.
- P5 204 “refexion index” à refractive index
Response: We are sorry for the typo mistake. We have corrected the typo as '' refractive index ''. Please see page 6, line 220.
- P5 L210: It’s difficult to discriminate well the absorption peak of MB because of the prominent extinction of gold nanoparticles. Moreover, absorption peak of MB is very variable depending on solvent polarity and hydrogen bond it can vary in presence of tannic acid and PEG.
Response: We do agree with the reviewer’s comment. Considering the complicated intermolecular interaction between Au@TNA nanoparticle and the functional groups of MB molecule, we tune down the relevant sentence as ''Notably, the absorption band position of Au@TNA NP and MB was overlapping at 600-650 nm. The further studies of the additional simulation and experiment designing works further study were necessary to examine the formation of J- or H-aggregation adsorption''. Please see page 6, lines 227-230.
- P5 L212-218: The presence of MB fluorescence during RAMAN experiment may also be induced by released free MB in solution. It’s very difficult (nearly impossible) to be sure that no MB is free in solution even after several purification cycles with centrifugation. Since fluorescence is very sensitive and MB is quite fluorescent very few free MB can lead to this result. Moreover, even well purified nanoparticles can slowly release MB.
Response: We thank for reviewer’s comment. We performed a fluorescence measurement of the supernatant solution from the 1-day aging time of Au@MB NPs in PBS solution at pH 7.4. About 2.4 % MB was released from the Au@MB NPs. The additional experiment result and relevant discussion have been added in the revised manuscript. Please see Figure S4, and page 6, lines 233-235.
- P6 Figure 2b in the caption there are two captions for figure 2b (fluorescence spectra or SERS measurement).
Response: We thank for reviewer’s question. We showed the lower and higher background signals by using 785 nm and 633 nm, respectively. The generation of the higher background signals was deduced due to the evolution of fluorescence from released MB by exciting at 633 nm. To make the story easier to follow, we have re-written the relevant discussion as ''……, strong fluorescence appeared to evolve the background signal, and negligible surface-enhanced Raman scattering peaks were obtained in the Raman spectrum (Figure 2b). ……To eliminate the typical fluorescence interference in the SERS spectra from the 633-excited MB molecules ……. Please see page 6, lines 232-236.
- P6 Singlet oxygen generation assay. I don’t understand why using RNO/imidazole, such a complex 1O2 probing system implies two successive reactions which can lead to errors. (https://doi.org/10.1007/s00216-019-01910-2). Moreover, RNO is a very reactive specie.
Response: We thank for reviewer’s comment. RNO and imidazole molecules are cheaper chemicals when compared with other singlet oxygen (1O2) indicator. As suggested by reviewer, we change the analysis method by replacing RNO/imidazole with ABDA indicator. ABDA is a popular indicator for determining the generation efficiency of 1O2 (ref. ACS Appl. Mater. Interfaces 2015, 7, 432−441 and Nat. Chem. Biol. 2011, 7, 504−511.), making the analysis results look more reliable. The additional measurement has been added in the revised manuscript. Please see Figure S5 and page 4, lines 142-144, and page 7, lines 246-248.
- The singlet oxygen quantum yield measurement method is wrong. There is no equation presented to explain the calculation of this quantum yield, although it is a quite complicated measurement. Methods for singlet oxygen measurement are described in other papers which use DPBF or water soluble anthracene derivatives. Some experimental considerations have to be taken. The consumption of the quencher has to be negligible to ensure that there is no change of quencher concentration during all experiment (indeed when the quencher concentration is lowered, its ability to react with singlet oxygen is lowered since singlet oxygen deactivate itself in the solution before reaching the quencher). Due to method errors, the singlet oxygen generation quantum yield by Au@TNA@MB is very over evaluated by comparison with MB alone.
Response: We thank for reviewer’s comment. Indeed, the deeper comparison of the difference between MB alone and Au@TNA@MB NPs is difficult in the current state and is beyond the scope of this paper. Considering the complicated issues to address photochemical and photophysical processes of MB-aggregated sate onto the surface of Au@TNA nanoparticles, we decided to remove the naked and inappropriate sentences. Please see page 7, lines 248-250. Furthermore, if possible, we may look for some potential collaboration with other groups to complete the quantification of 1O2 generation.
- P6 L237: Thermographic image are depicted in figure 3c and not a figure with MB photostability.
Response: We thank for reviewer’s comment. To make the description clearer, we re-written the sentences as ''……consistent heating temperature below 38 oC after 10 min of irradiation at 660 nm (Figure 3c). Noted that the absorption of Au@TNA@MB NPs showed a negligible change at 664 nm, indicating a better photostability …… '' Please see page 7, lines 251-253.
- P7 figure 4 c) it gives the impression that cells were treated during 4h under irradiation.
Response: We thank for reviewer’s comment. To prevent readers from confusing the PDT-treatment time, we re-written the relevant sentences and the corresponding figure caption. Please see page 8, lines 276-278 and the caption of Figure 4c.
Reviewer 2 Report
Nanoparticles for Enhanced Photodynamic Therapy of Bladder Cancer Cells” describes an interesting work in line with recent research on the conjugation of nanoparticles and PDT.
There are some minor issues that should be revised, for example:
N-hydroxysuccinimide, N,N-dimethyl-4-nitrosoaniline, N'- ethyl carbodiimide... N should be italic
Both designations: DI water and D.I. water are in the text, please uniformize
Page 3, an ~ 70% reduction rate...The authors means 70% reduction ratio? And also at page 8, line 285 ~ 70% cell death rate
The concentration of Gold appears subscript several times through the text, for example line 306, page 9 (10 ppm [Au]).
The major issue is about the procedure for the PDT experiments, the authors described “45 min, 2 h, and 4 h of coculture time was performed, followed by irradiation with a 660 nm laser (125 mW/cm2) for 8 min.”
The authors should explain if between the coculture with the drug and irradiation the culture media was removed and replaced by drug-free incubation media or not, because in the first case only the drug that the cells uptake will be active and in the second case the drug at the surface of the cells will be also active
Author Response
Reviewer 2
- N-hydroxysuccinimide, N,N-dimethyl-4-nitrosoaniline, N'- ethyl carbodiimide... N should be italic
Response: Thank you for the suggestion. We have revised the relevant text in the revised manuscript. Please see page 3, lines 112-113 and page 4, lines 148-149.
- Both designations: DI water and D.I. water are in the text, please uniformize
Response: Thank you for pointing this out. We have made the same representation as DI water in the revised manuscript.
- Page 3, an ~ 70% reduction rate...The authors means 70% reduction ratio? And also at page 8, line 285 ~ 70% cell death rate
Response: Thank you for the comment and suggestion. We have corrected the term ''rate '' as ''ratio '' in the revised manuscript. Please see in Page 3, line 135 and page 9, line 299.
- The concentration of Gold appears subscript several times through the text, for example line 306, page 9 (10 ppm [Au]).
Response: Thank you for pointing this out. We have made subscript expression for all the concentration of Gold in the revised manuscript.
- The major issue is about the procedure for the PDT experiments, the authors described “45 min, 2 h, and 4 h of coculture time was performed, followed by irradiation with a 660 nm laser (125 mW/cm2) for 8 min.”
The authors should explain if between the coculture with the drug and irradiation the culture media was removed and replaced by drug-free incubation media or not, because in the first case only the drug that the cells uptake will be active and in the second case the drug at the surface of the cells will be also active
Response: Thank you for the insightful comments. In these PDT experiment groups, the particle-including culture media was removed and then replaced by fresh culture medium (drug-free and particle-free condition). To clarify these points, we have re-written the relevant in Experiment Section. Please see in Page 4, lines 165-167.
Reviewer 3 Report
This manuscript reported by Yi-Chun Chiu and coworkers entitled Development of Folic Acid-conjugates and methylene Blue-adsorbed Au@TNA Nanoparticles for Enhanced photodynamic Therapy of Bladder Cancer Cells reports the synthesis, structural characterization of Au@TNA nanoparticles and the photodynamic assays against cancer cell lines. The results obtained clearly show that the prepared NPs have potential to be use as photosensitizers, with the presence of folic acid making the difference.
The introduction of this manuscript is very well written, describing the state of the art of PDT, the advantageous and disadvantageous of porphyrinic derivatives as photosensitizers and the need to prepare other type of noncytotoxic materials as gold nanoparticles. The synthesis of gold nanoparticles by assisted reduction of tannic acid is quite cleaver, and few papers on polyphenols assisted reduction of HAuCl4 have been reported on this type of synthesis. The authors should introduce up to two references concerning earlier studies.
The characterization performed on the Au@TNA NPs was so carefully conducted, that it lacks a characterization discussion for Folic Acid-conjugates. please add a structural characterization and a brief discussion to the manuscript or to the supporting information
Since Au@TNA NPs consisted of spherical shape and triangular shape NPs, did the authors considered the influence of the shape on the photodynamic activity of the conjugates? Please add a comment related to this subject.
Regarding the literature, perhaps there is a newer reference to replace reference 44 from 1990.
Author Response
Reviewer 3
- The synthesis of gold nanoparticles by assisted reduction of tannic acid is quite cleaver, and few papers on polyphenols assisted reduction of HAuCl4 have been reported on this type of synthesis. The authors should introduce up to two references concerning earlier studies.
Response: Thank you for the suggestion. We have added two literature reports related to the polyphenol-assisted reaction to fabricate Au nanoparticles for the Introduction, as suggested by Reviewer. Please see Introduction Section, lines 94-97 with reference 31 and 32.
- The characterization performed on the Au@TNA NPs was so carefully conducted, that it lacks a characterization discussion for Folic Acid-conjugates. Please add a structural characterization and a brief discussion to the manuscript or to the supporting information
Response: Thank you for the valuable comment. We performed additional examinations of FA-conjugated Au@TNA@MB NPs with TEM, Uv-visible spectra, and zeta potential measurement. The new results have added in the revised manuscript. Please see Figure S7. These relevant sentences have also been added in the text. Please see page 9, lines 308-310 and page 10, 311-314.
- Did the authors considered the influence of the shape on the photodynamic activity of the conjugates? Please add a comment related to this subject.
Response: Thank you for the insightful question. The size- and shape-dependent treatment is an important aspect. The previous studies mostly focus on the efficacy of endocytosis with drug concentration. Theoretically, the size of 40-60 nm is better for endocytosis and 100-400 nm is better for enhanced permeability and retention (ref. ACS Nano 2015, 9, 9, 8655–8671 and Bioconjugate Chem. 2016, 27, 10, 2225–2238). Another study suggests that rod-like Iodo-borondipyrromethene (IBDP) nanoparticles could be internalized by cancer cells more and possess a better imaging ability compared to the corresponding spherical micelles (ref. Zhang, J. Rod-like BODIPY nanomaterials with enhanced photodynamic activity. New Journal of Chemistry 2020.). Still, there are limited discussion about shape-dependent PDT efficacy to date. The aspect provided us about the new design concept of PDT NPs platform in the future. Thank you for the useful comment and we have added a brief discussion in Conclusion Section in revised manuscript. Please see page 10, lines 338-341.
- Regarding the literature, perhaps there is a newer reference to replace reference 44 from 1990.
Response: We thank for reviewer’s comment. We have replaced the original ref 44 with newer one. Please see reference 43.
Round 2
Reviewer 1 Report
A lot of work has been done by authors and additional experiments have been carried out.
In general, this publication is well written, but in several paragraphs formulations should be corrected as some sentences are difficult to understand.
For example:
P6: 227: Notably, the absorption band position of Au@TNA NP and MB was overlapping at 600-650 nm
--> the absorption band positions ... ...were overlapping
P6 228: The further studies of the additional simulation and experiment designing works further study were necessary to examine the formation of J-or H-aggregation adsorption
--> too complicated.
--> additional simulations and experiments are necessary to examine the potential formation of ....
P6 231: Upon laser excitation at 633 nm, strong fluorescence appeared to evolve the background signal,
--> reformulation required
P7 248: The close adsorption of MB onto Au NPs would gently decreased 1O2 generation due to the energy quenching process by transformation from the excited-state MB molecule to the surface of the Au NPs.
--> reformulation required
P7 252: Noted that the absorption of Au@TNA@MB NPs showed a negligible change at 664 nm (Figure 3a), which indicating a better photostability, while the corresponding absorption of the MB-alone group remarkably decreased with irradiation time (Figure S6).
--> reformulation required
In the conclusion paragraph there are several problems:
L 321: The Au@TNA NPs could carry massive MB molecules with a high production yield of 1O2 generation upon excitation at 650 nm.
--> I don't understand this sentence. MB is not a massive molecule (MW = 320g/mol <500g/mol), so I guess it is a massive amount of MB featuring high production yield of 1O2
L 325: the targeted activated photocytotoxicity --> the target activated photocitotoxicity
L 335: retain --> retains
L 340: which more likely to be uptake by the cells
Author Response
Reviewer 1
- P6: 227: Notably, the absorption band position of Au@TNA NP and MB was overlapping at 600-650 nm
--> the absorption band positions ... ...were overlapping
Response: Thank you for the suggestions. In the revision, “Notably, the absorption band position of Au@TNA NP and MB were overlapping at 600-650 nm.” Please see P6 227.
- P6 228: The further studies of the additional simulation and experiment designing works further study were necessary to examine the formation of J-or H-aggregation adsorption
--> too complicated.
--> additional simulations and experiments are necessary to examine the ....
Response: Thank you for the suggestions. In the revision, “Additional simulations and experiments are necessary to examine the potential formation of J-or H-aggregation adsorption” Please see P6 228 to 230.
- P6 231: Upon laser excitation at 633 nm, strong fluorescence appeared to evolve the background signal,
--> reformulation required
Response: Thank you for the suggestions. In the revision, “Strong fluorescence appeared and negligible surface-enhanced Raman scattering peaks were obtained in the Raman spectrum under laser excitation at 633 nm (Figure 2b).” Please see P6 231 to 232.
- P7 248: The close adsorption of MB onto Au NPs would gently decreased 1O2 generation due to the energy quenching process by transformation from the excited-state MB molecule to the surface of the Au NPs.
--> reformulation required
Response: Thank you for the suggestions. In the revision, “When the excited-state MB molecule transformed and adsorbed to the surface of the Au NPs, the energy quenching process would gently decreased 1O2 generation.” Please see P7 247 to 249.
- P7 252: Noted that the absorption of Au@TNA@MB NPs showed a negligible change at 664 nm (Figure 3a), which indicating a better photostability, while the corresponding absorption of the MB-alone group remarkably decreased with irradiation time (Figure S6).
--> reformulation required
Response: Thank you for the suggestions. In the revision, “Compared to MB-alone group (Figure S6), the absorption of Au@TNA@MB NPs showed a negligible change at 664 nm (Figure 3a), which indicated better photostability of Au@TNA@MB than MB-alone.” Please see P7 251 to 253.
In the conclusion paragraph:
- L 321: The Au@TNA NPs could carry massive MB molecules with a high production yield of 1O2 generation upon excitation at 650 nm.
--> I don't understand this sentence. MB is not a massive molecule (MW = 320g/mol <500g/mol), so I guess it is a massive amount of MB featuring high production yield of 1O2
Response: Thank you for the suggestions. In the revision, “The Au@TNA NPs could carry massive amount of MB molecules featuring high production yield of 1O2 generation upon excitation at 650 nm.” Please see P10 327 to 328.
- L 325: the targeted activated photocytotoxicity --> the target activated photocitotoxicity
Response: Thank you for the suggestions. In the revision, “the target activated photocytotoxicity”. Please see P10 331.
- L 335: retain --> retains
Response: Thank you for the suggestions. In the revision, “but retains a very low dark toxicity to normal cells”. Please see P10 333.
- L 340: which more likely to be uptake by the cells
Response: Thank you for the suggestions. In the revision, “we expected changing the size and the shape of Au@TNA nanoparticles to enhance the affinity to cancer cells would offer us a promising future design to improve delivery efficiency of PDT NPs.”. Please see P10 338
