Upconversion nanocomposite for programming combination cancer therapy by precise control of microscopic temperature

Combinational administration of chemotherapy (CT) and photothermal therapy (PTT) has been widely used to treat cancer. However, the scheduling of CT and PTT and how it will affect the therapeutic efficacy has not been thoroughly investigated. The challenge is to realize the sequence control of these two therapeutic modes. Herein, we design a temperature sensitive upconversion nanocomposite for CT-PTT combination therapy. By monitoring the microscopic temperature of the nanocomposite with upconversion luminescence, photothermal effect can be adjusted to achieve thermally triggered combination therapy with a sequence of CT, followed by PTT. We find that CT administered before PTT results in better therapeutic effect than other administration sequences when the dosages of chemodrug and heat are kept at the same level. This work proposes a programmed method to arrange the process of combination cancer therapy, which takes full advantage of each therapeutic mode and contributes to the development of new cancer therapy strategies.

Remarks to the Author: The present work aims at optimizing combined cancer therapy (chemotherapy, C T, and photothermal therapy, PTT) by designing a clever nanosystem including a nanothermometer (upconverting lanthanide-based nanoparticle, UC NP), a chemical drug (doxorubicin), and a photothermal agent (palladium(II) phthalocyanine). The main finding of the study lies in demonstrating that sequential application of the two treatments (C T and PTT) has a far better efficiency than simultaneous application as it is usually practiced. The trick here is to monitor the temperature of the cells via the UC NP, excited at 980 nm), which subsequently allows one to tune the light fluence and irradiation dose at 730 nm to generate C T, and then PTT. The advantage of the sequential treatment is first demonstrated in vitro on MIA PaC a-2 cells and finally in vivo on Balb/c mice. Additionally, another important finding is that generation of heat shock protein (HSP) is much reduced in the sequential treatment as opposed to the simultaneous protocol, meaning that lower dosage of chemodrug and photothermal agent can be used, reducing side effects. All data are reported with standard deviations and satisfying statistics is provided for the in vivo experiments (5 mice for each of the cohorts). Other experimental procedures are described with necessary details, in particular the morphology of the nanocomposites. This contribution represents an important advance in the field of cancer therapy and will have a broad impact in both the biomedical and photophysics communities. C onclusions are convincing and at this stage, further experiments are not required, as the principle is well established. It will of course have to be checked for each specific case (cancer type, nature of chemodrug and photothermal agent) but this is clearly out of the scope of such a communication. As a conclusion, I recommend publication after some polishing of the manuscript (and, also some clarifications).
-For instance the introduction is somewhat long with repetitions, the role of the two wavelengths used (980 for T measurements and 730 nm for therapy, with low -C T -and high -PTT -power densities) should be described in a clearer way. -C aption to Fig. 3. Please give Er(III) concentration and, also, excitation wavelength -There is probably an error in reporting power densities used for the in vitro study (46 and 140 W/cm2) in the text (lines 207, 209), while corresponding captions to figures mention mW. -There is some confusion in the description of the in vitro experiments. While at the beginning (lines 229-230) is it clearly stated that all cell experiments were conducted with MIA PaC a-2 cells, Figs. 5c/d report images of HeLa cells (as said in the caption). If different cell lines were used, please explain why and, also, clearly state this in the captions to Fig. 5.
-C aption to Figure 5. Please explain the meaning of 730 nm (-) and 730 nm (+) - Figure 7c. Please give the dosages of drug and photothermal agent used -Line 352. C an the authors quantify what they mean by "less dosages"? Which proportion? Getting this information would strengthen the article. Some suggested text improvements -Line 35, replace "received" with resulted in" -Line 39, replace "nanocomposite" with "a nanocomposite" -Line 42, replace "have" with "has" -Line 85, replace "a n on-doping" with "an undoped" J.-C . Bünzli

Comments:
In this manuscript, the authors, using upconversion luminescencent technology to monitor the microscopic temperature (eigen temperature) of nanocomposite, come up with the novel concept of programming combination cancer therapy by precise control of microscopic temperature, in which lower laser power density is used for chemotherapy drug release while a higher one is used for photothermal therapy. The rational experiment design, solid data, and significant conclusion will contribute to the rapid development of cancer treatment in the future, which is definitely of interest and value to the chemistry and nanomaterial communities. In my opinion, this manuscript should be considered for acceptance after minor modifications. My comments are as follows: Response: We greatly appreciate the carefully reading and the positive comment from the reviewer. Response: We greatly appreciate this valuable advice. TGA analysis is indeed a commonly used method to determine the composition of nanomaterials. However, TGA analysis is not suitable to be used here for characterizing the nanocomposite in this work since TR-UCNS has multiple components (PdPc, DPPC and DOX) and the percentage of each component is hard to be told by weight loss. We are thankful to the reviewer for helping us improve our work by raising this suggestion. As an alternative solution, the approximate number of PdPc and DOX in each nanoparticle can be clearly given by computing the data in Supplementary Figure 3d and 3e.
The diameters of NaLuF 4 :20%Yb,2%Er and Please see the added contents below, "According to the data shown in Figure S3d and S3e, the molar of PdPc loaded in 20 mg YSUCNP is 1.28 µmol (2 mg×76.4%×1197.8 g mol -1 ).  implied that high initial temperature as used in conventional combination therapy method will cause significant increase of heat shock protein, while the generation of HSP70 will reduce the therapeutic effect of combination therapy which is shown in Supplementary

As a conclusion, I recommend publication after some polishing of the manuscript (and, also some clarifications).
Response: We greatly appreciate the carefully reading and the positive comment from the reviewer. The corresponding polishing and clarifications are listed below as requirement by the reviewer's further comments.

CT -and high -PTT -power densities) should be described in a clearer way.
Response: We greatly appreciate this valuable advice from the reviewer. The introduction part in the revised manuscript was amended to reduce repetitive descriptions. Meanwhile, in the second paragraph of introduction, we clarified the roles of 980 nm and 730 nm lasers and supplemented the detailed process of programmed combinational therapy.
Revised parts of the introduction section are labeled in yellow in the manuscript. Fig. 3. Please give Er(III) concentration and, also, excitation wavelength Response: Thanks for this helpful advice. We indicated the concentration of Er(III)

Comment 2. Caption to
(2.5×10 -5 mol L -1 ) and the excitation wavelength (980 nm) for luminescence spectra comparisons in Fig. 3a. However, programmed combination therapy achieved a more complete killing effect with only 1.3 % cancer cells survived. This indicates that programmed combination therapy uses less drug and heat to realize an ideal killing effect that conventional combination therapy may reach by using more drug and heat. If the same therapeutic effect as programmed combination therapy, up to 8 folds of chemodrug is needed (20 µM of doxorubicin) or up to 2.6 folds of 730 nm laser power density is needed (400 mW cm -2 ).
The supplemented explanation in Discussion section is shown below and is labeled in yellow in revised manuscript, "When the dosages of chemodrug and heat are kept at low level (2.5 µM of DOX and heat generated by ~150 mW cm -2 of 730 nm laser), programmed combination therapy can achieve 39 folds improvement in therapeutic effect in vitro than conventional combination therapy that initiates chemotherapy and photothermal therapy at the same time. This indicates that programmed combination therapy uses less drug and heat to realize an ideal killing effect that conventional combination therapy may reach by using more drug and heat. It is worth noting that if the same therapeutic effect as programmed combination therapy is wanted, up to 8 folds of DOX is needed (20 µM) or up to 2.6 folds of 730 nm laser power density is needed (400 mW cm -2 ) ( Figure S7a and S7b)." Comment 8. Some suggested text improvements -Line 35, replace "received" with resulted in" -Line 39, replace "nanocomposite" with "a nanocomposite" -Line 42, replace "have" with "has" -Line 85, replace "a n on-doping" with "an undoped" J.

-C. Bünzli
Response: We appreciate the advices for improving our manuscript. The suggested words for replacement have been added in the manuscript and highlighted in yellow. We are sincerely grateful to the reviewer for the recognition of our work as well as the comments to improve the manuscript.

Point-by-Point Response to Reviewers
Reviewer #1 (Remarks to the Author): The Response: We greatly appreciate the reviewer for this valuable suggestion and apologize for the miscalculation of the volume of nanoparticles. We corrected the equation and presented the calculating steps in a clearer way. The calculating processes are shown below.
We carefully checked other calculations and confirmed that they are correct. The corrections to the numbers of PdPc and DOX molecules do not affect the results of other parts of the manuscript.