Near-infrared light and tumor microenvironment dual responsive size-switchable nanocapsules for multimodal tumor theranostics

Smart drug delivery systems (SDDSs) for cancer treatment are of considerable interest in the field of theranostics. However, developing SDDSs with early diagnostic capability, enhanced drug delivery and efficient biodegradability still remains a scientific challenge. Herein, we report near-infrared light and tumor microenvironment (TME), dual responsive as well as size-switchable nanocapsules. These nanocapsules are made of a PLGA-polymer matrix coated with Fe/FeO core-shell nanocrystals and co-loaded with chemotherapy drug and photothermal agent. Smartly engineered nanocapsules can not only shrink and decompose into small-sized nanodrugs upon drug release but also can regulate the TME to overproduce reactive oxygen species for enhanced synergistic therapy in tumors. In vivo experiments demonstrate that these nanocapsules can target to tumor sites through fluorescence/magnetic resonance imaging and offer remarkable therapeutic results. Our synthetic strategy provides a platform for next generation smart nanocapsules with enhanced permeability and retention effect, multimodal anticancer theranostics, and biodegradability.


Response to the comments from the editor and three referees
Response to the editor The comments: The reports seem to be quite clear, and we will naturally need you to carefully address all of the issues raised. We would recommend that you expand your discussion of similar size changing systems in the literature and highlight the differences to your system. In addition we would ask that you carefully consider the laser power being used in your experiments. The current safety standard is 0.3 W/cm 2 , as a minimum we need you to confirm that the ethical approval for your animal work approved the use of the higher laser powers.

Our response and revision:
Thanks for your precious comments, evaluation and publication recommendation of our manuscript. The "size changing systems" reported in our study is quite different from already reported systems. It is well known that the design and development of sophisticated nanocapsules for targeted delivery of theranostic agents to solid tumors hold great promise for improving the treatment efficacy and minimizing the systemic toxicity (J. Am. Chem. Soc. 2019, 141, 4406-4411;J. Am. Chem. Soc. 2017, 139, 4584-4610;Macromolecules, 2013, 46, 9169-9180). Despite tremendous potential, currently most researches who studies nanocapsules like structures, have mainly focused on a single stimuli responsive system, which does not meet precise control and initiation of such responsive systems (Angew. Chem. Int. Ed. 2019, 58, 159-163;Angew. Chem. Int. Ed. 2018, 57, 17048-17052;J. Am. Chem. Soc. 2017, 139, 7522-7532;J. Am. Chem. Soc. 2014, 136, 14896-14902;Mol. Pharmaceutics 2014, 11, 1599-1610. Moreover, the metabolism of nanocapsules in biological environment has rarely been studied (J. Am. Chem. Soc. 2018, 140, 4666-4677;Langmuir 2016, 32, 6211-6225). Unfortunately, most of the existing nanocapsules lack the simultaneous combination of the multiple therapeutic/imaging modalities into one platform to synergistically enhance therapeutic efficacy or to obtain theranostic nanomaterials (ACS Appl. Mater. Interfaces 2019, 11, 1886-1895Acc. Chem. Res. 2015, 48, 2935-2946. More importantly, the toxicity of most of the materials used to construct nanocapsules is not approved by FDA. Therefore, it is difficult for these nanocapsules to enter clinical trials. In our study, we have constructed an intelligent NIR/TME dual-responsive nanocapsule (made of DOX-ICG@Fe/FeO-PPP) for enhanced tumor accumulation and improved therapy efficacy. This research work includes the following novelty: 1. Under the stimulation of NIR light and acid TME, the nanocapsules could shrink and decompose into small-sized nano-drugs, accompanied by drug release.
2. Meanwhile, the nano-drugs enter into tumors and overproduce the reactive oxygen species (ROS) by synergistic catalysis of Fenton reaction based on Fe/FeO NCs and light activation from ICG, which relieves hypoxic condition and promotes the synergistic therapy of tumors.

3.
In vivo experiments demonstrated that these nanocapsules can offer remarkable imaging and therapeutic results.
4. The materials that make up nanocapsules are basically FDA certified (PLGA, PEG and ICG). The furthermore experimental results show that the biosafety of the produced nanocapsules is relatively reliable.
Hence, this study presents the design of smart nanocapsules with enhanced tumor accumulation, highly effective therapy and diagnosis to accelerate exploitation and clinical translation of intelligent theranostics` nanocapsules.
To ensure that our research work conforms to the ethical approval for our animal work, we reduced the laser power to 0.3 W/cm 2 . In addition, we improved the encapsulation of ICG by PLGA-PEG-PNIPAM (The coating amount changed from 2 mg to 3 mg). Finally, the feasibility of this laser power (808 nm, 0.3 W/cm 2 ) has been proved both in vitro and in vivo. Following experimental results were included in revised manuscript:

Response to the referee 1:
The comments: This manuscript reported the synthesis and preparation of near-infrared (NIR) light-/tumor microenvironment (TME)-dual responsive size-switchable nanocapsules, which not only shows NIR light and TME responsive size changes for enhanced tumor accumulation, but also can regulate the unfavorable TME for improving the synergistic therapy effect of cancer. The synthesis and nanocapsules structure is relatively new. The characterizations are overall comprehensive and the results have good scientific value and novelty. Therefore, I suggest the acceptance for publication after the following points are addressed:

Our response:
Thanks to the respected reviewer for appreciating comments and positive evaluation on our manuscript. We have addressed all the questions of reviewer and tried our best to perform additional experiments where needed. Q1. In this manuscript, for the first time, the authors synthesized nanocapsules that could simultaneously perform photodynamic and chemodynamic therapy. Because both ROS treatment modes need to consume hydrogen peroxide, is there competition in the treatment process?

Our response:
Thanks a lot to the reviewer's professional question. Actually, the role of photodynamic therapy in our treatment methods is relatively weak. The main purpose of ICG and laser is to promote the shrinkage of nanocapsules. The laser power used is relatively low and the consumption of hydrogen peroxide is limited.
Q2. In the ESR spectra of Fe/FeO NCs and ICG@Fe/FeO-PPP nanocapsules with DMPO, the author should give the physical parameters represented by abscissa.

Our response and revision:
Many thanks to the reviewer for pointing out this short coming. We have revised  Q3. Can Fe/FeO NCs catalyze the release of oxygen from hydrogen peroxide in tumor microenvironment?

Our response:
Fe/FeO NCs catalyze the release of oxygen from hydrogen peroxide in tumor microenvironment. According to our proposed reaction mechanism, Fe/FeO NCs mainly undergo the above-mentioned Fenton reaction process in the tumor microenvironment. In this condition, Fe/FeO NCs will dissociate and produce Fe 3+ species. These Fe 3+ have been proved to catalyze the decomposition process of hydrogen peroxide to produce oxygen in tumor cells (Nat. Nanotechnol., 2007, 2, 577-583;Anal. Chem., 2012, 84, 5753-5758).
Q4. In order to explain the formation and assembly process of nanocapsules more clearly, the authors should describe the infrared spectra in detail in the supporting information.

Our response and revision:
Thanks a lot to the reviewer for this valuable suggestion. We have elaborated the infrared spectra in detail and revised the supporting information discussion on page 4 line 10-15.
"The formation of PPP was confirmed by the FTIR spectrometer, which was marked in Many thanks to the reviewer raising this question. The efficiency of Fe 2+ in catalyzing fenton reaction is higher than that of other valence states. Hence, the core-shell structure of Fe/FeO NCs helps to obtain more Fe 2+ in tumor microenvironment. Because the oxidation of Fe in the core is a process from low to high valence, Fe 2+ can be directly obtained from shell ferrous oxide in weak acid tumor microenvironment. So the selection of Fe/FeO NCs is very meaningful.

Response to Referee Two Reviewer Comments:
After reading carefully the manuscript entlited "Near-Infrared Light and Tumor Microenvironment Dual Responsive 2 Size-Switchable Nanocapsules for Multimodal Tumor Theranostics" by Zhiyi Wang, Yanmin, Zeeshan Ali, Hui Yin, Fugeng Sheng, Jian Lin, Baodui Wang and Yanglong Hou, I would not suggest its publication in Nature Communications.
The reason of my decision is due to the lack of novelty of the presented work. In this manuscript, the authors prepare a multifunctional system that can be used for diagnosis and cancer treatment, and in order to do that the combine many different elements like magnetic nanoparticles, block copolymer (BTW, the FDA has approved PLGA for some uses but PNIPAM, the thermo-responsive polymer is not approved), DOX and ICG as building blocks for creating hybrid nano-capsules of 200 nm in diameter. All the experiments are well described and many of the results sounds, other like the EPR capacity of 200 nm nanocapsules is a fairy tale.
Furthermore, a theoretical pH value of 5.4 within solid tumour is not possible, (this value is not compatible with the cell viability). However, the main problem of this manuscript is that there are many other multifunctional systems that have been already published and many of them combine similar elements to produce a theragnostic system like the presented in this work. In addition, the presented manuscript isn't a break-through in the topic, is a good manuscript that present nice results for being published elsewhere like ACS applied materials and interfaces or Nano-Research, but not in Nature Communication.

Our response and revision:
Thanks to the reviewer for critical comments and evaluation on our manuscript. It is well known that the design and development of sophisticated nanocapsules for targeted delivery of theranostic agents to solid tumors hold great promise for improving treatment efficacy and minimizing systemic toxicity (J. Am. Chem. Soc. 2019, 141, 4406-4411;J. Am. Chem. Soc.2017, 139, 4584-4610;Macromolecules, 2013, 46, 9169-9180). Despite tremendous potential, currently most researches on nanocapsules are mainly focused on single stimuli responsive, which does not meet precise control and initiation of the responsive systems (Angew. Chem. Int. Ed. 2019, 58, 159-163;Angew. Chem. Int. Ed. 2018, 57, 17048-17052;J. Am. Chem. Soc. 2017, 139, 7522-7532;J. Am. Chem. Soc. 2014, 136, 14896-14902;Mol. Pharmaceutics 2014, 11, 1599-1610. Moreover, the metabolism of nanocapsules in biological environment has rarely been studied (J. Am. Chem. Soc. 2018, 140, 4666-4677;Langmuir 2016, 32, 6211-6225). Furthermore, most of the existing nanocapsules lack the simultaneous combination of the multiple therapeutic/imaging modalities into one platform to synergistically enhance therapeutic efficacy or to obtain theranostic nanomaterials (ACS Appl. Mater. Interfaces 2019, 11, 1886-1895Acc. Chem. Res. 2015, 48, 2935-2946. More importantly, the toxicity of the most of the materials used to construct nanocapsules is not approved by FDA. Therefore, it is difficult for these nanocapsules to enter clinical trials. In our study, we for the first time have constructed an intelligent NIR/TME dual-responsive nanocapsule (made of DOX-ICG@Fe/FeO-PPP) for enhanced tumor accumulation and improved therapy efficacy. This research work includes the following novelty: 1. Under the stimulation of NIR light and acid TME, the nanocapsules could shrink and decompose into small-sized nano-drugs, accompanied by drug release.
2. Meanwhile, the nano-drugs enter into tumors and overproduce the reactive oxygen species (ROS) by synergistic catalysis of Fenton reaction based on Fe/FeO NCs and light activation from ICG, which relieves hypoxic condition and promotes the synergistic therapy of tumors.

3.
In vivo experiments demonstrated that these nanocapsules can offer remarkable imaging and therapeutic results.
4. The materials that make up nanocapsules are basically FDA certified (PLGA, PEG and ICG). The furthermore experimental results show that the biosafety of the produced nanocapsules is relatively reliable.
Hence, this study presents the design of smart nanocapsules with enhanced tumor accumulation, highly effective therapy and diagnosis to accelerate exploitation and clinical translation of intelligent theranostics nanocapsules.
In addition, we have responded to other mentioned questions and performed experiments or revisions to improve our manuscript as follows: The reviewer pointed out that the "block copolymer (BTW, the FDA has approved PLGA for some uses but PNIPAM, the thermo-responsive polymer is not approved)". This misunderstanding was caused by the sentence "It worths mentioning here that these nanocapsules has potential clinical application as the constituent materials of these have already been approved by the US FDA" in our manuscript.
Actually, PEG and PLGA in the PLGA-PEG-PNIPAM which was synthesized in our manuscript have passed FDA certification, except for PNIPAM. Molecular weight of PLGA, PEG and PNIPAM in our research was about 15000, 4000 and 2000 respectively. We have tried to reduce the proportion of PNIPAM in this polymer system. We are deeply sorry for our imprecise expression. Thanks a lot for the reviewer pointing it out. We have removed this sentence to make the discussion clearer in our manuscript.
Subsequently, the reviewer also mentioned "DOX and ICG as building blocks for creating hybrid nano-capsules of 200 nm in diameter. All the experiments are well described and many of the results sounds, other like the EPR capacity of 200 nm nanocapsules is a fairy tale".
Hence, in our study, we synthesized nanocapsules with initial size of 200 nm to provide long-lasting blood circulation and less clearance of organs. But when the nanocapsules reach the tumor tissues, they could shrink and decompose into small-sized nanodrugs triggered by photothermal effect of ICG under laser irradiation and lower pH value in TME (Figure 4c The reviewer's prospective "However, the main problem of this manuscript is that there are many other multifunctional systems that have been already published and many of them combine similar elements to produce a theragnostic system like the presented in this work. In addition, the presented manuscript isn't a break-through in the topic, is a good manuscript that present nice results for being published elsewhere like ACS applied materials and interfaces or Nano-Research, but not in Nature Communication" is highly expected to change after considering above revisions and the fact that, our research system is significantly different from other reported systems.
(3) The materials that make up nanocapsules are basically FDA certified (PLGA, PEG and ICG). The furthermore experimental results show that the biosafety of the produced nanocapsules is relatively reliable.
(4) We have also explored the metabolic problems of the nanocapsules after cancer treatment, which is of great significance against the background of the great challenge of nano-biomedicine.
Following above changes and explanations, we strongly believe that the reviewer will reconsider to recommend our work for Nature Communications.

Response to Referee Three Reviewer Comments:
This study reports a novel kind of near-infrared (NIR) light and tumor microenvironment (TME) dual responsive size-switchable nanocapsules (DOX-ICG@Fe/FeO-PPP). The smartly engineered DOX-ICG@Fe/FeO-PPP nanocapsules were shown to not only shrink and decompose into small-sized nanodrugs upon drug release, but also regulate TME to overproduce reactive oxygen species (ROS) for enhanced synergistic therapy in tumors. Sufficient experiments were performed to verify the magic theranostic performance of DOX-ICG@Fe/FeO-PPP nanocapsules. This paper is also well-written and well-organized. Therefore, I would like to recommend this paper for publication in Nature Communications after addressing the following issues:

Our response and revision:
We are thankful to the reviewer for positive comments and evaluation on our manuscript. We responded every comment from the referee one by one.  Inter. 2015, 7, 14896-14904;J. Am. Chem. Soc. 2013, 135, 835-843). We have introduced the mechanisms for the synthesis of the PLGA-PEG-PNIPAM (PPP).
Water-oil ratio (Vdichloromethane/Vwater) for the initial emulsion and PVA are two key conditions in the synthesis of nanocapsules. Water-oil ratio for the initial emulsion  Q3. Where is the source of 1 O2? Why does the 1 O2 generation rely on pH? It seems that the Fenton reaction of ICG@Fe/FeO-PPP can only generate •OH rather than 1 O2.

Our response:
Thanks a lot to the reviewer's professional comment. There are two sources of 1 O2, one is the oxygen in tumor microenvironment, and the other is the air entering the cavity of nanocapsules during the synthesis of nanocapsules. The change in acidity of the system will affect the stability of nanocapsules, which will cause the amount of 1 O2 generation.
Q4. The generated •OH amount should be measured by using more accurate quantitative methods, such as methylene blue (MB)   Q7. There is no data to show the advantage of laser-trigged shrinkage of DOX-ICG@Fe/FeO-PPP nanocapsules in facilitating the deep tumor tissue penetration of nanocapsules.

Our response and revision:
We again thank the reviewer for pointing out another important aspect. Due to the spatial resolution of MRI, we tested real-time MRI of KB tumor-bearing mice Q8. Some in vitro cell experiment results are suggested to be removed from supplementary information to article. Also, please add statistical analysis to Figure 4a, 4b, 5d, 6c, S16b, S20.

Our response and revision:
Thanks a lot to the reviewer for this kind suggestion. We removed the mentioned results from supplementary information and the corresponding statistical analysis results have been added to the Figure 4a, 4b, 5d, 6c, S16b and S20 in revised manuscript.
Q9. The quality of the figures should be improved. Currently the words in the figures are not shown clearly.

Our response and revision:
We are thankful to the reviewer for this kind suggestion. Wherever necessary, the text font size was increased and we did our best to improve the resolution of figures according to the requirements of this journal. In addition, we have made it certain that the text in all figures of our manuscript is clearly readable. These corrections were highlighted in the revised manuscript.