Photo-Cleavable Polycations-Wrapped Upconversion Nanoparticles for Efficient siRNA Delivery and Cancer Therapy
Round 1
Reviewer 1 Report
In this manuscript, the authors developed a photo-cleavable polymercationic wrapped upconversion nanoparticles (PC-UCNPs) for spatially and temporally controllable siRNA delivery. PC-UCNPs was synthesized by in situ reversible addition−fragmentation chain transfer (RAFT) polymerization of photo-cleaved 5-(2-(dimethylamino)ethoxy)-2-nitrobenzyl acrylat (DMNA) monomer and poly(oligo(ethylene oxide)acrylate (PEG) mononer through a chain transfer agent that anchored on the surface of silica coated upconversion nanoparticles (UCNPs@SiO2), The synthesized PC-UCNPs showed high loading capacity of negatively charged siRNA, and the outer layer coating of HA not only targeted cancer cell but also protected siRNA from degradation. Both in vitro and in vivo experiments confirmed good therapeutic effects of PC-UCNPs/siRNA/HA. The experiment results could support conclusion, and I would like to recommend for publication in Targets after minor revisions:
1. The authors mentioned in Figure 3a that highly cationic surface of PC-UCNPs/HA and PC-UCNPs/siRNA/HA contributed to their permeabilization, therefore the zeta potential of PC-UCNPs should be characterize.
2. I suggest the author to add semi-quantitative analysis of Figure 4b.
3. I suggest to extend the experiment period to 48 h for PC-UCNPs/siRNA/HA stability verification experiment in Figure S9.
4. To verify biological safety of PC-UCNPs/siRNA/HA, MTT alone is not enough. I suggest the authors to supplement hemolysis test.
Author Response
- The authors mentioned in Figure 3a that highly cationic surface of PC-UCNPs/HA and PC-UCNPs/siRNA/HA contributed to their permeabilization, therefore the zeta potential of PC-UCNPs should be characterize.
Response: We thank the reviewer for the valuable suggestion. According to the reviewer’s suggestion, we have measured the zeta potential of PC-UCNPs as 58 ± 2.2 mV and the corresponding result has been added in Figure S8 and Page4 line 116-117.
- I suggest the author to add semi-quantitative analysis of Figure 4b.
Response: According to the reviewer’s suggest, we have carried out semi-quantitative analysis for FAM florescence in Figure 4b using Image-Pro Plus 6.0, and the corresponding results have been added in Figure S10 and Page7 line 199-202.
- I suggest to extend the experiment period to 48 h for PC-UCNPs/siRNA/HA stability verification experiment in Figure S9.
Response: According to the reviewer’s suggest, we have extended the experiment period to 48 h for PC-UCNPs/siRNA/HA stability experiment, and the corresponding result have been supplemented in Figure S9.
- To verify biological safety of PC-UCNPs/siRNA/HA, MTT alone is not enough. I suggest the authors to supplement hemolysis test.
Response: We thank the reviewer for the valuable suggestion. According to the reviewer’s suggest, we have carried out hemolysis test for PC-UCNPs/siRNA/HA, and the corresponding results have been added in Figure S12 and Page8 line 243-245, Page 13 line 472-481.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript He et al describes interesting and potent nanoparticles for the delivery of siRNA into tumor cells in vitro and in vivo. The nanoparticles are built up from lanthanide-doped nanoparticles coated with a SiO2 layer and finally with a functional polymer coating. By implementing a photo-cleavable linker between the siRNA-binding group and the main polymer chain, a fast and efficient siRNA release can be induced upon NIR-radiation. This mechanism addressed two major opposing goals, the efficient siRNA binding and protection and the release at the site of action. Furthermore, for many hard nanoparticles used for nucleic acid delivery, they are adsorbed onto the surface, which often lead to degradation or desorption after contact with biological fluids (eg. serum). For circumventing this problem, the authors coated the siRNA-loaded nanoparticles with hyaluronic acid (HA). In their work this final layer protected the siRNA against degradation/desorption and by using HA a targeting moiety was introduced for enhanced cellular uptake via the CD44 receptor. In addition, the HA coating reduces the zeta potential towards negative values which will probably extend the circulation time after systemic administration.
The authors characterized the most important physicochemical properties of the nanoparticles. The siRNA loading, the stability and the stimuli release upon NIR radiation and hyaluronidase-mediated degradation of the HA layer was nicely demonstrated in solution and in vitro using HepG2 cancer cells. Finally, the biological effects after delivering an apoptosis-inducing siRNA (siPLK1) in vitro and in tumor-bearing mice underlined the potency of these nanoparticles.
However, there are some issues and questions which need to be addressed prior to accepting this manuscript.
First of all, in the text are supplementary figures mentioned but they were not included. This must be forwarded as they are important data for the manuscript.
See:
L 89 Fig. S1
L 92 Fig. S2
L 95 Fig. S3-5
L 96 Fig. S6
L 100 Fig. S7
L 118 Fig. S8
L 136 Fig S9
L 241 Fig. S10
In L 100 Fig S7 should show a GPC measurement and the molecular weight of the polymer was determined to be 11.6kDa. How was this measurement performed? It is not listed in the experimental part and more important, was the measurement done with the nanoparticle or a reference polymer without?
For Fig. S7 the increase of the zeta potential after methylation is mentioned and makes sense. Do the authors know if the quaternization is necessary for efficient siRNA binding? In most cases the permanent cationic charge leads to increased cytotoxicity. And here is another point which I hardly believe: that is the not existing cytotoxicity of their nanoparticles. Unfortunately, the dose-dependent viability assay is missing. And, all functional assays (Fig 5) were performed after 24h. It is nice to see that these nanoparticles are able to downregulate the mRNA, protein levels so fast and showing high apoptosis rates specifically upon radiation and just for the PLK1 siRNA! However, I would like to see some more data for later time points (48-72h). At later time points, it is possible that the nanoparticles may decompose and generate toxic effects. Another aspect is that the nanoparticles rupture the lysosomal membrane very efficiently and this should not be toxic to cells? In my experience, polymers which damage endo/lysosomal membranes so strong also damage the cells to some extent. Even the stimuli responsiveness upon radiation was demonstrated only for early time points (see Fig 4, 4h). A closer look into the “tightness” of the nanoparticles at later time points would be important for evaluating the biocompatibility for further in vivo applications. In this study, I know, only the radiated group has shown the anti-tumor effect of the treatment. But the siRNA-loaded nanoparticles were only injected intratumorally and not systemically.
In the experimental section, the authors need to describe some information more in detail:
What was the exact buffer composition used for siRNA loading? Also, the typical volumes and masses of the compounds used for a loading/complexation procedure need to be listed! What was the final mass ratio, 1:1 or higher? For the HA coating, a detailed description of this procedure is completely missing and must be included as well!
For the CLSM experiments: What were the amounts of nanoparticles/siRNA in these studies?
The discussion of their results must be extended. Beside the introduction and some references in the results and discussion part for describing some facts, are their findings not really discussed.
And generally, the image sharpness is quite poor and must be improved.
Author Response
- In the text are supplementary figures mentioned but they were not included. This must be forwarded as they are important data for the manuscript. See: L 89 Fig. S1; L 92 Fig. S2; L 95 Fig. S3-5; L 96 Fig. S6; L 100 Fig. S7; L 118 Fig. S8; L 136 Fig S9; L 241 Fig. S10.
Response: We are sorry for the careless mistake. The supplementary Figures have been uploaded with the revised manuscript.
- In L 100 Fig S7 should show a GPC measurement and the molecular weight of the polymer was determined to be 11.6kDa. How was this measurement performed? It is not listed in the experimental part and more important, was the measurement done with the nanoparticle or a reference polymer without?
Response: We thank the reviewer for the valuable suggestion. GPC measurement was performed with polystyrene as reference polymer. The corresponding measurment process has been supplemented According to the reviewer’s suggest, we have supplemented the detailed measurement process in the information material in P11, line 415-421.
- For Fig. S7 the increase of the zeta potential after methylation is mentioned and makes sense. Do the authors know if the quaternization is necessary for efficient siRNA binding? In most cases the permanent cationic charge leads to increased cytotoxicity. And here is another point which I hardly believe: that is the not existing cytotoxicity of their nanoparticles. Unfortunately, the dose-dependent viability assay is missing.
Response: We thank the reviewer for the valuable suggestion. The quaternization is necessary for efficient siRNA binding since the low zeta potential of UCNPs@SiO2-polymer before quaternization (-2.1 ± 1.1 mV, in Figure S8) could not load negatively charge siRNA efficiently. Negatively charged HA was wrapped over positively charged PC-UCNPs, and gave zeta potential of -23 ± 2.6 mV, therefore the final product PC-UCNPs/siRNA/HA didn’t demonstrate cytotoxicity. The dose-dependent viability assay has been supplemented as Figure S11, and showed about 87% of cell viability even with 200 μg/mL PC-UNCPs/siRNA/HA. The corresponding discussion has been supplemented in Page 7, line 241-243.
- All functional assays (Fig 5) were performed after 24h. It is nice to see that these nanoparticles are able to downregulate the mRNA, protein levels so fast and showing high apoptosis rates specifically upon radiation and just for the PLK1 siRNA! However, I would like to see some more data for later time points (48-72h). At later time points, it is possible that the nanoparticles may decompose and generate toxic effects.
Response: We thank the reviewer for the valuable advice. According to the reviewer’s suggestion, we have supplemented mRNA and PLK1 protein expression levels, and cell viability tests for PC-UCNPs/siPLK1/HA treated HepG2 cells at 48 h and 72 h respectively after treatments. The experiment results are as follows (Figure R1), which showed similar results as that of 24 hours post treatment.
Figure R1. Gene silencing and cell viability. (a) PLK1 mRNA expression levels via qRT-PCR, (b) PLK1 protein expression via ELISA and (c) Relative HepG2 cell viability after HepG2 cells was treated with PBS and PC-UCNPS/siRLK1/HA for 48 h or 72 h. The data error bars indicate means ± S.D. (n = 5)
- Another aspect is that the nanoparticles rupture the lysosomal membrane very efficiently and this should not be toxic to cells? In my experience, polymers which damage endo/lysosomal membranes so strong also damage the cells to some extent. Even the stimuli responsiveness upon radiation was demonstrated only for early time points (see Fig 4, 4h). A closer look into the “tightness” of the nanoparticles at later time points would be important for evaluating the biocompatibility for further in vivo applications. In this study, I know, only the radiated group has shown the anti-tumor effect of the treatment. But the siRNA-loaded nanoparticles were only injected intratumorally and not systemically.
Response: We thank the reviewer for bringing this important point. After endosomal escape, the positively charted polymer coating was cleaved into non-toxic small molecular compounds, which would metabolized fast and wouldn’t generate cellular toxicity. According to the reviewer’s suggestion, we have continuously measured zeta potential and size of PC-UCNPs/siRNA/HA at 4 h and 24 h after HAase treatment. The experiment result is as follows (Figure R2), which showed particle structure stability. Considering experiment performance convenience, we chose intratumoral administration of PC-UCNPs/siRNA/HA in this manuscript, and we don’t expect its systemic toxicity considering its little effect on cell viability (Figure S11) and low hemolysis percentage (Figure S12).
Figure R2. Zeta potential and DLS analysis of PC-UCNPs/siRNA/HA at 4 h and 24 h after HAase treatment.
- In the experimental section, the authors need to describe some information more in detail: What was the exact buffer composition used for siRNA loading? Also, the typical volumes and masses of the compounds used for a loading/complexation procedure need to be listed! What was the final mass ratio, 1:1 or higher? For the HA coating, a detailed description of this procedure is completely missing and must be included as well! For the CLSM experiments: What were the amounts of nanoparticles/siRNA in these studies?
Response: We thank the reviewer for the valuable suggestion. According to the reviewer’s suggest, the buffer composition used for siRNA loading has been added in Page 12 line 417-418. The volumes and masses of siRNA and siPLK1 used for loading/complexation procedures have been added in Page 13 line 444, 445, 450, 466, 467. The final mass ratio for PC-UCNPs/siRNA is 1:1. The detailed description for HA coating has been added in Page 12 line 422-425. For CLSM experiment, 5 µg/mL nanoparticles (with 10 nM siRNA loading) were incubated with HepG2 cells, corresponding description have been added in Page 13 line 442, 448, 454 Page 12 line 450, 456, 461 and figure captions of Figure 3, 4.
- The discussion of their results must be extended. Beside the introduction and some references in the results and discussion part for describing some facts, are their findings not really discussed.
Response: According to the reviewer’s suggestion, we have supplemented corresponding discussions of PC-UCNPs zeta potential at Page 4, line 116-117; semiquantitative analysis of intracellular FAM-siRNA fluorescence at Page 6, line 199-202; hemolysis percentage measurement and discussion at Page 7, line 243-245.
- The image sharpness is quite poor and must be improved.
Response: According to the reviewer’s suggestion, we have replaced all the figures with improved image quality and larger image size of 600 dpi.
Author Response File: Author Response.pdf
Reviewer 3 Report
#1 What is the deffinition of polymercationic? In my opinion authors should use term polycations or reffering to polymer-DNA construct - polyplexes
#2 Please check if the abbreviation suits monomer name correctly, e.g. polymerization of photo-cleaved 5-(2-(di- 16 methylamino)ethoxy)-2-nitrobenzyl acrylate (DMNA) and poly(oligo(ethylene oxide)acrylate is not PEG
#3 The authors refer to supplementary materials that have not been made available
#4 Why authors used P(PEG-co-DMNA) with molecular mass equal 11.6 kDa? What was theoretical molecular mass and the most important dispersity index value for this polymer?
English language is fine.
Author Response
- What is the definition of polymercationic? In my opinion authors should use term polycations or referring to polymer-DNA construct – polyplexes.
Response: We thank the reviewer for pointing out the spelling mistake. We have replaced “Polymercationic” with “polycations” in manuscript title; abstract; Page 2, line 49; Page 11, line 401, 415; Page 14, line 534 of the revised manuscript.
- Please check if the abbreviation suits monomer name correctly, e.g. polymerization of photo-cleaved 5-(2-(di- methylamino)ethoxy)-2-nitrobenzyl acrylate (DMNA) and poly(oligo(ethylene oxide) methyl ether acrylate is not PEG.
Response: We thank the reviewer for pointing out these mistakes. We have re-abbreviated photo-cleaved 5-(2-(di- methylamino)ethoxy)-2-nitrobenzyl acrylate as MENA and poly(oligo(ethylene oxide) methyl ether acrylate as OEMA in the revised manuscript.
- The authors refer to supplementary materials that have not been made available.
Response: We are sorry for the careless misstate, we have uploaded supplementary information with revised manuscript.
- Why authors used P(PEG-co-DMNA) with molecular mass equal 11.6 kDa? What was theoretical molecular mass and the most important dispersity index value for this polymer?
Response: P(PEG-co-DMNA) (renamed as P(OEMA-co-MENA) in revised manuscript) (11.6 kDa) demonstrated good loading capacity of siRNA. Its theoretical molecular mass has been calculated as 18.5 kDa via n= Mtheory/Mn equation (n is the degree of polymerization, Mtheory is the theoretical molecular weight of polymer, Mn is the repeat unit molecular weight of polymer), and PDI is calculated as 1.18 (Figure S7).
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The authors sufficiently addressed all my questions and remarks.
Author Response
We thank the reviewer for us work approbation.
