ROS-augmented and tumor-microenvironment responsive biodegradable nanoplatform for enhancing chemo-sonodynamic therapy

Journal Pre-proof ROS-augmented and tumor-microenvironment responsive biodegradable nanoplatform for enhancing chemo-sonodynamic therapy Jie An, Yong-Guo Hu, Kai Cheng, Cheng Li, Xiao-Lin Hou, Gang-Lin Wang, XiaoShuai Zhang, Bo Liu, Yuan-Di Zhao, Ming-Zhen Zhang PII:

S0142-9612(20)30007-7

DOI:

https://doi.org/10.1016/j.biomaterials.2020.119761

Reference:

JBMT 119761

To appear in:

Biomaterials

Received Date: 6 November 2019 Revised Date:

2 January 2020

Accepted Date: 4 January 2020

Please cite this article as: An J, Hu Y-G, Cheng K, Li C, Hou X-L, Wang G-L, Zhang X-S, Liu B, Zhao YD, Zhang M-Z, ROS-augmented and tumor-microenvironment responsive biodegradable nanoplatform for enhancing chemo-sonodynamic therapy, Biomaterials (2020), doi: https://doi.org/10.1016/ j.biomaterials.2020.119761. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Tumor-Microenvironment Biodegradable Nanoplatform for

ROS-Augmented

Responsive

and

Enhancing Chemo-Sonodynamic Therapy Jie An1, Yong-Guo Hu1, Kai Cheng1, Cheng Li1, Xiao-Lin Hou1, Gang-Lin Wang1, Xiao-Shuai Zhang1, Bo Liu1, Yuan-Di Zhao1,2,*, Ming-Zhen Zhang3,*

1

Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for

Optoelectronics-Hubei Bioinformatics & Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, P. R. China 2

Key Laboratory of Biomedical Photonics (HUST), Ministry of Education, Huazhong University of

Science and Technology, Wuhan 430074, Hubei, P. R. China 3

Institute of Medical Engineering, School of Basic Medical Science, Health Science Center, Xi’an

Jiaotong University, Xi’an 710061, Shanxi, P. R. China

*Co-corresponding author.

Tel/Fax:

+86-27-8779-2202.

Email

address:

[email protected] (M.Z. Zhang).

1

[email protected]

(Y.D.

Zhao);

Abstract: Nanocarrier for augmenting the efficacy of reactive oxygen species (ROS) by tumor microenvironment (TME) has become an emerging strategy for cancer treatment. Herein, a smart biodegradable drug delivery nanoplatform with mitochondrial-targeted ability, pH-responsive drug release and enzyme-like catalytic function is designed. This efficient ROS-generating platform uses ultrasound with deeper penetration capability as excitation source for combined chemotherapy and sonodynamic therapy (SDT) of tumor. In vitro experiments show that the nanoplatform can co-load Ce6 and DOX and be degraded in slight acid environment, and the DOX release rate is 63.91±1.67 %. In vivo experiments show that the nanoplatform has extremely biosafety and can be enriched in tumor site and excluded from body after 24 h. More significantly, after combined treatment, the tumors are eliminated and the mice still survive healthily without recurrence after 60 d. This is because not only it can achieve mitochondrial targeting and use platinum particle to increase oxygen content in TME to enhance the effect of SDT, but also it can use weak acidic TME to accelerate drug release to achieve the combination of chemotherapy and SDT. The probe provides a new strategy for designing ROS-based nanoplatform for the treatment of malignant tumor. Keywords: pH-responsive; tumor hypoxia; mitochondria targeting; chemotherapy; sonodynamic therapy

2

1. Introduction Reactive oxygen species (ROS) is one group of ubiquitous active molecules in human body, but its existence is a double-edged sword [1]. Low level ROS can be used as the second messenger of physiological regulation, however, excessive ROS will exceed the antioxidative capacity of cell and cause its death [2, 3]. Cancer cell is so overwhelmingly dependent on antioxidant that it is more vulnerable to exogenous ROS or compound that destroys the antioxidative system [4, 5]. Therefore, elevating ROS level in cancer cell has been considered as an effective strategy to eradicate the cell, which has been widely used in recent years. With the deepening of work, researchers have found that the therapeutic effect of ROS on cancer cell is unsatisfactory, and there are still some key issues should be solved urgently. Firstly, the extremely short lifespan of ROS limits its diffusion distance (<20 nm), and it is often removed before it plays its role [6]. Secondly, the ROS production mainly depends on oxygen molecule at tumor site, however, it is known that most solid tumors are in hypoxic state due to limited oxygen diffusion or unstable blood flow in tumor microvascular, which is not conducive to the generation of ROS [7]. Furthermore, the traditional ROS generation relies on light or X-ray excitation, but the penetration depth and transmission efficiency of light is limited by tissue and X-ray radiation often leads to some harm to body [8, 9]. Finally, the therapeutic effect of single ROS on tumor is restricted, and combining with chemotherapy, immunotherapy or other treatments is indispensable to enhance the treatment efficacy [10, 11]. In order to overcome these difficulties, researchers have explored various ROS-generating platforms for ROS treatment. To enhance the therapeutic effect, some nanocarriers have been used to directly deliver ROS-generating reagents to mitochondria, who is vulnerable to ROS, by modifying the targeting mitochondria molecules (e.g. polypeptide and small molecules) on

3

the surface [12-14]. This is because mitochondria is vital cellular organelle not only to supply energy for cell, but also control the intrinsic pathway of apoptosis [15]. Some nanoplatforms have been also loaded with H2O2 catalysts (e.g., catalase [16], MnO2 [17] and Pt nanoparticle [18]) to catalyze the decomposition of endogenous high content H2O2 (100 µM~1 mM) to oxygen in the tumor tissue, and alleviate the limitation of hypoxia on ROS treatment [19]. To overcome the depth limitation and safety problem of traditional ROS generation, ultrasound, an external excitation source with deep penetration capability, is also used in sonodynamic therapy (SDT) [20]. Nevertheless, the design of nanoplatform that can overcome these difficulties at the same time and combine chemotherapy is still challenging. Hollow nanomaterials (e.g., hollow gold nanosphere [21], hollow silica [22] and hollow manganese dioxide [23]) have been widely used in drug/gene delivery due to their high drug loading capacity. Dopamine (DA), as a bioactive molecule, exists widely in living organism [24]. Due to its good biocompatibility, easy polymerization and surface modification (via Michael addition or Schiff base reaction with thiol or amine), it has captured much attention in recent years [25]. In this paper, a biodegradable hollow polydopamine nanoparticle (P@HP) embedded with platinum nanoparticle (Pt) was prepared, after co-load with doxorubicin (DOX) and chlorine e6 (Ce6), and modification with triphenylphosphonium (TPP), a mitochondrial-targeting molecule, ultrasound with deep penetration was used as excitation source for this efficient ROS-generating platform to combine chemotherapy and SDT of tumor (Fig. 1). The nanoprobe (CDP@HP-T) had a great deal of unique advantages as novel smart ROS-augmented nanocomposite. Firstly, CDP@HP-T could be enriched in the tumor site through enhanced permeability and retention effect of solid tumor. Secondly, as a pH-responsive nanoprobe, it could release drug after biodegradation in weakly acidic tumor microenvironment

4

(TME) and/or lysosomal compartment. The released DOX killed tumor cell by inhibiting the cellular DNA replication. Concomitantly, Pt embedded CDP@HP-T could act as the catalase-like catalyst to trigger the decomposition of tumor endogenous H2O2 for in situ generation of O2, alleviating the hypoxia of tumor site and further enhancing the therapeutic efficacy of SDT. Subsequently, nanoprobe could carry drug to target mitochondria by TPP, and further enhancing the therapeutic efficacy. Ultimately, the probe achieve the combination of chemotherapy and SDT. In vitro and in vivo experiments demonstrated that the probe had perfect biocompatibility, it could effectively relieve the hypoxic state of the tumor, resulting in a remarkable enhanced therapeutic effect of combined chemotherapy and SDT to eliminate tumor. This probe offers a new idea for improving the traditional ROS treatment in the future.

Fig. 1. Schematic illustration of the synthesis route of CDP@HP-T and chemo-sonodynamic combined therapy.

5

2. Results and Discussion 2.1. Synthesis and characterization of CDP@HP-T. P@HP was prepared by zeolitic imidazolate framework 8 (ZIF-8) doped with Pt as intermediate. Frist of all, the uniform Pt-embedded ZIF-8 nanoparticle (P@ZIF-8) was prepared by zinc nitrate and 2-methylimidazole and doped with polyvinylpyrrolidone (PVP) modified Pt (Fig. 2A). Then, according to the characteristic that the binding ability of Zn2+ to dopamine was stronger than it to 2-methylimidazole, the latter in P@ZIF-8 was gradually replaced via chelation competition induced polymerization in the presence of excessive dopamine, thus P@HP with uniform size was formed by the chelation of Zn2+ to dopamine [26]. The formation of P@HP was further observed by TEM. After 30 min of reaction, small amorphous particles could be observed on the surface of the nanostructure at P@ZIF-8 (Fig. S1), comparing with their original smooth. A ‘core–shell’ nanostructure was formed and the ‘core’ was gradually decreased as the reaction was prolonged, while the PDA shell became thicker and stronger. After 6 h, the ‘core’ completely disappeared and finally formed hollow P@HP. Since polydopamine could serve as anchor for the resultant metal (0), Pt was well dispersed and evenly distributed on the shell of hollow particle [24, 27]. Elemental mapping revealed that Pt, Zn, N and O elements were distributed on the surface of probe, but their contents in central area were low, demonstrating the probe had hollow structure (Fig. 2A). Furthermore, the structure of the product was analyzed by the X-ray diffraction (XRD). It was found the diffraction patterns of P@ZIF-8 agreed well with that of pure ZIF-8 and four additional specific peaks at high angles of 30°~90°, corresponding to the (111), (200), (220), and (311) of Pt. As for P@HP, the diffraction peaks of Pt remained while that of ZIF-8 disappeared, which was replaced by scattering diffraction of the amorphous polymer shell (Fig. S2). The experimental results showed that hollow dopamine

6

was finally formed. Moreover, it was also found that there were irregular pores with diameter below 10 nm on the surface of P@HP (Fig. S1). The nitrogen adsorption-desorption isotherms of P@HP (Fig. S3A) were detected, it presented representative type-IV curves with capillary condensation step at P/P0=0.2~0.6, which was typically associated with uniform mesoporous. Moreover, a capillary condensation step hysteresis loop occurred in the P/P0 range of 0.8~1.0, corresponding to a relatively broad pore size distribution in the range of 2~10 nm and a peak pore size of 1.8 nm (Fig. S3B). The BET surface area and total pore volume, analyzed by the nitrogen physisorption were 170.8 m2/g and 0.329 cm3/g respectively. At last, attributing to the coordination between three carboxyl groups in Ce6 and metal ion (Zn2+) on the P@HP, Ce6 could load on the P@HP [28]. As the special structure of polydopamine, DOX could also be loaded on the P@HP via π-π stacking. The DOX and Ce6 were co-loaded in P@HP, and SH-PEG2000-TPP was modified on the surface of polydopamine by Michael addition reaction to obtain the final probe (CDP@HP-T). The result of transmission electron microscopy (TEM) showed that CDP@HP-T had hollow structure with quite uniform size at ~250 nm and shell thickness at ~50 nm. X-ray photoelectron spectroscopy analysis confirmed that the feature peaks of CDP@HP-T coincided with those of Pt alone (Fig. 2B). The high resolution spectrum of Pt 4f peak revealed that Pt0 was dominant in CDP@HP-T. These results proved that hollow polydopamine embedded with Pt had been successfully synthesized. In addition, as the stretching vibration of amide I band at 1633 cm-1 in SH-PEG2000-TPP was confirmed by fourier transform infrared, it was indicated that the carboxyl group of TPP and amino group of SH-PEG2000-NH2 were successfully linked by amidation reaction (Fig. 2C), which established the ground for mitochondrial targeting of the probe.

7

Fig. 2. Transmission electron microscopy (TEM) and magnification of P@ZIF-8 and CDP@HP-T, element mapping for CDP@HP-T (A); X-ray photoelectron spectroscopy of P@PVP and CDP@HP-T, insert: Pt 4f region (B); infrared spectra of modified material before and after targeting molecular coupling (C).

8

Next, in order to study whether CDP@HP-T had pH-responsive decomposition behavior, the probe was dispersed in phosphate buffer (PBS) at pH 5.5, 6.5 and 7.4 for 4 h, respectively. As shown by TEM, CDP@HP-T remained intact at pH 7.4, while the varying degrees of degradation was occurred in mildly acidic conditions (Fig. S4). Decreasing pH value, the disintegration degree of CDP@HP-T increased, indicating that the probe could be degraded in acidic TME (~pH 6.5) [29]. For the mechanism of the biodegradation, in the previous reports [24, 30], it was found when the pH value of solution decreased from 8.5 to 5, the thickness of deposited polydopamine film gradually decreased, it showed that accompanying with the increase of hydrogen proton owing to the decrease of pH, a series of chemical equilibriums would shift towards the degradation of product. In this work, because the CDP@HP-T was mesoporous structure, it had a huge surface area, which would also accelerate its degradation in acidic tumor microenvironment. Moreover, the pH-biodegradable performance of polydopamine has also been observed in other previous studies [31-33]. Based on the pH-response characteristic, the capability of CDP@HP-T as pH-responsive drug carrier was further studied. The absorption peaks of DOX (490~500 nm) and Ce6 (404 and 660 nm) were all observed in absorption spectrum of CDP@HP-T (Fig. 3A), indicating the probe successfully co-loaded Ce6 and DOX. The loading contents of Ce6 and DOX increased with the rise of adding Ce6 and DOX. At feed ratio of drug and P@HP-T was 6, the loading of Ce6 and DOX reached to a rather high level of 61.65±1.18 % and 65.03±1.82 %, respectively (Fig. 3B). It was manifested that the probe could co-load Ce6 and DOX for combination therapy.

9

Fig. 3. The ultraviolet-visible absorption spectra of Ce6, DOX, P@HP and CDP@HP-T (A); Ce6 and DOX loading content in CDP@HP-T at different feeding drug:P@HP ratios (B); Ce6 (C) and DOX (D) controlled release curves of CDP@HP-T triggered by different pH; the change of dissolved oxygen concentration after CDP@HP-T reacting with different concentrations of H2O2 (E); the electron spin resonance (ESR) spectra of CDP@HP-T before and after US (F); the ultraviolet-visible absorption spectra of DPBF and CDP@HP-T mixed solution with US time (G); reactive oxygen species production by CDP@HP-T under different conditions (H); the change of hydrated particle size and PDI of CDP@HP-T (I).

10

The drug release behavior of Ce6 and DOX from CDP@HP-T was then studied in solutions at different pH values. Compared with pH 7.4, the release rate in acidic solution increased significantly with the decrease of pH (Fig. 3C and D), owing to pH-responsive decomposition of CDP@HP-T. It was found that Ce6 could be released more in acid PBS solution (Fig. S5). Approximately 74.79±2.82 % loaded Ce6 and 63.91±1.67 % loaded DOX were released from CDP@HP-T after incubation at pH 5.5 for 8 h, much higher than those at pH 6.5 and 7.4. Consequently, the pH-responsive CDP@HP-T could be used to release drugs in acidic TME to achieve combined therapy. As some pioneering studies had confirmed, the hypoxic status of solid tumor might limit the therapeutic effect of SDT on tumor [34]. Considering the tumor tissue general contained high level of H2O2, Pt presented significant catalase-like activity to induce the decomposition of H2O2 to O2, therefore, the catalase-like activity of CDP@HP-T was tested. It was found that the dissolved O2 level was stable in blank H2O2 solution, while rapid O2 generation to reach a stable level was observed after CDP@HP-T was added (Fig. 3E). Moreover, CDP@HP-T exhibited obvious concentration-dependence on H2O2 decomposition, the higher dissolved O2 level was obtained with the increase of H2O2 concentration. Then, UV-vis spectra of remainder H2O2 were recorded after different reaction conditions to verify the catalase-like activity of CDP@HP-T (with Pt). It was found that the concentration of H2O2 (10 mM) was negligible change after ten US irradiation cycles and reaction with CD@HP-T (without Pt) (Fig. S6A and B). As the relatively low frequency, power of ultrasound, the degradation of H2O2 by ultrasound was limited. In contrast, the concentration of H2O2 was decreased along with the reaction time in the sample containing CDP@HP-T (Fig. S6C). All of these results confirmed Pt nanoparticles could obviously catalyze the decomposition of H2O2.

11

As ensuring oxygen supply would increase ROS generation, the ability of CDP@HP-T to produce ROS

under

ultrasonic

excitation

was

further

tested

by

1

O2

capture

agent

2,2,6,6-tetramethylpiperidine (TEMP) and ROS agent 1,3-diphenylisobenzofuran (DPBF), respectively. In experiment, weak triplet ESR signals were observed in TEMP+CDP@HP-T group (Fig. 3F), this might be due to the fact that Ce6 was also a photosensitizer and the whole experimental process was unavoidably exposed to light. It was also found that the poor ESR signals strength of TEMP+H2O2+US group was close to that of TEMP+CDP@HP-T group. This might be due to the fact that ultrasound irradiation could also produce a small amount of 1O2. However, after ultrasonic treatment, these signals were obviously enhanced, indicating that the probe had sonodynamic effect. Interestingly, the strongest ESR signals were presented in the system containing H2O2 (200 µΜ), this was because CDP@HP-T could catalyze H2O2 decomposition to O2, increasing the oxygen content in the system and then producing more 1O2. The absorption peak intensity of DPBF was decreased with the increase of ultrasonic time (Fig. 3G) because the furan ring in DPBF was destroyed by ROS and DPBF was consumed continuously with the increase of ultrasonic time. In addition, the changes of DPBF absorption under different treatment conditions showed that, the loss of DPBF absorption in probe group mixed with H2O2 after ultrasound treatment (US) was significantly higher than those in other control groups (Fig. 3H), indicating that CDP@HP-T could trigger H2O2 decomposition to O2, and more 1O2 was produced after US, which was consistent with ESR result. Based on the above experiment, it showed that CDP@HP-T could be used to alleviate hypoxia in tumor microenvironment and enhance ROS level in sonodynamic process. The stability, biocompatibility and biodegradability of CDP@HP-T were also discussed. The results showed that the size and polydispersity index (PDI) of CDP@HP-T did not change obviously

12

in ultrapure water after one week (Fig. 3I). In hemolysis assay, when the probe concentration was as high as 2 mg·mL-1, the hemolysis rate was still less than 5 %, which was within the acceptable range of intravenous administration (Fig. S7) [35]. The biodegradability of nanocarrier delivery system played an essential role in the metabolism of probe. To evaluate the biodegradability of CDP@HP-T, the nanocomposite was dispersed in simulated body fluid (SBF). TEM showed the probe was degraded gradually over time (Fig. S8), indicating that CDP@HP-T could be slowly degraded in vivo and excreted from the body with high biological safety, which provided a possibility for further application in model animal. 2.2. In vitro experiment with CDP@HP-T. In the design of probe, CDP@HP-T should target mitochondrial, accordingly, the intracellular localization of probe with or without TPP was observed by confocal laser scanning microscopy. In experiment, green fluorescence corresponded to mitochondria stained with Mito Tracker Green marker, red fluorescence corresponded to Ce6, and blue fluorescence corresponded to the nuclei stained with Hoechst 33342. It was found Ce6 fluorescence in cell incubated with CP@HP-T presented overlap with Mito Tracker after 4 h, and the merged fluorescence tended to yellow (Fig. 4A). In contrast, Ce6 and Mito Tracker in cell incubated with CP@HP (without TPP) showed less colocalization. Quantitative result showed the fluorescence overlap coefficient of the former was 0.82±0.04, while that of the latter was 0.59±0.02 (Fig. 4B), indicating a significant difference between them (p<0.01). Pearson’s colocalization coefficient (Rr) was also used to determine the colocalization of these two fluorescence signals. It was found that Rr was 0.61±0.08 for CP@HP-T and only 0.19±0.05 for CP@HP, suggesting the specific intracellular targeting of mitochondria by nanoprobe with TPP (p<0.01).

13

Fig. 4. Confocal image of subcellular localization of CP@HP-T and CP@HP after 4 h (A); the statistic of Pearson’s correlation coefficient and overlap coefficient (Rr) for A (B); intracellular hypoxia imaging using [Ru(dpp)3]Cl2 as indicator (C); quantification of intracellular fluorescence intensity in C (D); ROS production detected by fluorescence of DCFH-DA in 4T1 cell (E); quantification of intracellular fluorescence intensity in E (F); **: p<0.01, ***: p<0.001.

14

Next, the ability of probe for alleviating the intracellular hypoxia and enhancing ROS generation was evaluated. In experiment, cell was treated with deferoxamine, a hypoxia-mimetic agent, to simulate hypoxia in solid tumor. Intracellular hypoxia was detected with [Ru(dpp)3]Cl2, an intracellular O2 level indicator, its red fluorescence would be enhanced with the decrease of O2 level. The result showed that the red fluorescence of hypoxia group was distinctly stronger than that of normoxia group (Fig. 4C), while p<0.01 (Fig. 4D); but there was no significant change in the intracellular fluorescence of after incubation with Pt-free HP-T for 4 h in both groups. Interestingly, compared with the control and Pt-free HP-T groups, the intracellular fluorescence were significantly decreased after P@HP-T treatment (p<0.001) in both groups; however, the fluorescence in hypoxia group was still significant stronger than that in normoxia group (p<0.01), indicating nanoprobe containing Pt could effectively elevate intracellular O2 content. After that, the enhancement of ROS production

by

CDP@HP-T

was

examined.

Intracellular

ROS

was

detected

using

2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), its green fluorescence would be strengthened with the increase of ROS in cell. The result showed that there was only sporadic green fluorescence in the cell of CDP@HP-T group if without US and in the control group with US (Fig. 4E), and there was no significant difference between them (Fig. 4F), which might be due to a little of endogenous ROS existing in cell. As expected, the cells treated with CDP@HP-T and CD@HP-T after irradiation with US showed bright green fluorescence, while the former was significantly stronger than the latter (p<0.01). This was because Ce6, a sonosensitizer, could produce ROS under US, and CDP@HP-T probe containing Pt could catalyze the decomposition of endogenous H2O2 to O2, elevating the source of ROS generation; while Pt-free CD@HP-T could not. These data revealed that CDP@HP-T probe could effectively increase the level of ROS generation in cell, which was extremely beneficial

15

to enhance the effect of SDT.

Fig. 5. Confocal image of 4T1 cell after incubation with DP@HP-T at different pH values (7.4 and 6.5) for 2 h (A); cell viabilities of 3T3 and 4T1 cells incubated with P@HP-T at different concentrations (B); cell viabilities of 4T1 cell treated with varied probes after US (C); calcein-AM and PI stained fluorescence imaging of 4T1 cell treated with varied probes after US (D); quantification of intracellular fluorescence intensity in D (E); cell viabilities of 4T1 cell treated with varied probes (2 µg·mL−1 Ce6) at pH 6.5 for 4 h and then US (F);*: p<0.05, **: p<0.01, ***: p<0.001.

16

As the pH-responsive decomposition of probe, the release behavior of DP@HP-T probe without Ce6 was also investigated under simulated acidic TME. Under confocal fluorescence imaging, the intracellular DOX red fluorescence in pH 6.5 medium was obviously stronger than that in neutral medium (Fig. 5A). This was because the degradation of DP@HP-T in acidic environment increased the release of DOX, and it was mainly concentrated in the cytoplasm owing to the short incubation time. This confirmed that the probe could be used as an acidic TME response drug delivery nanoplatform. Based on the above observation, the combined therapy efficiency of probe was evaluated. First, the cytotoxicity of nanoplatform was measured by MTT assay. The survival rates of mouse embryonic fibroblast (3T3) and breast cancer cell (4T1) were still higher than 90% even when the probe concentration reached to 200 µg·mL-1 (Fig. 5B), indicating that the material itself exhibited negligible cytotoxicity to cell. Moreover, the combined chemotherapy and SDT based on CDP@HP-T was also evaluated. It was found that US had only slight cytotoxicity to blank cell (Fig. 5C), and CP@HP-T, CDP@HP and CDP@HP-T themselves had little cytotoxicity if without US. However, the toxicity of probe to tumor cell increased evidently after US, and the killing effect was positively correlated with the concentration of probe. As for CP@HP-T without DOX and CDP@HP groups, the cell viabilities were similar. This was due to the targeting effect of TPP in CP@HP-T, which enabled the probe to specifically target mitochondria and increase the toxicity of probe to cell. And the CDP@HP group could also increase the toxicity of probe to cell by DOX slowly released from probe. Furthermore, the cell viability of CDP@HP-T with DOX was slightly lower than that of CP@HP-T without DOX and CDP@HP groups. Compared with these groups without ultrasound treatment, free DOX possessing brilliant anticancer ability because small molecules could easily diffuse into cells (p<0.01). When the concentration was 2 µg·mL-1 (Ce6 equivalent), the survival rate

17

in single SDT group (CP@HP-T+US, 51.16±8.01 %) was significantly lower than that before US (p<0.001), while the survival rate in combined treatment group (CDP@HP-T+US, 37.08±3.56 %) was also lower than that before US (p<0.001) (Fig. 5C). The result also showed that the probe had better combined effect of chemotherapy and SDT than single therapy (p<0.05). Interestingly, the therapeutic effect of mitochondrial targeted probe group (CDP@HP-T+US) was apparently higher than that of non-targeted probe group (CDP@HP+US) (p<0.05), and their difference was more significant at higher concentration (3 µg·mL-1) (p<0.01). This was due to the targeting effect of TPP, which enabled the probe to specifically target mitochondria and enhance the killing effect on tumor cell. The calcein-AM (AM, green fluorescence) and propidiumiodide (PI, red fluorescence) were further used to co-stain living and dead cells respectively in order to observe the enhanced therapeutic effect of mitochondrial targeting (Fig. 5D). It was found both cells only treated by US or CP@HP-T emerged large area of green fluorescence instead of red fluorescence, explaining that their killing effect on cell could be neglected. However, non-targeting SDT group (CP@HP+US) exhibited apparently strong red fluorescence in partial cell, and the targeting probe (CP@HP-T+US) showed stronger red fluorescence in almost all cell after SDT treatment (most of the cell in this group die. The fluorescence quantitative analysis demonstrated that the therapeutic effect of mitochondrial targeted probe group (CP@HP-T+US) was apparently higher than that of non-targeted probe group (CP@HP+US)，and there existed obviously significant differences between them (p<0.01). (Fig. 5E). These quantitative analyses were consistent with the results of the picture presentation. It was suggested that targeting could enhance the killing effect of SDT on cell, because more targeting probe was aggregated on mitochondria resulting in stronger killing effect. This result was consistent with MTT result. To further study the effect of pH on cell therapy, 4T1 cell was

18

incubated with different probes (2 µg·mL-1, Ce6 equivalent) at pH 6.5 for 4 h. Compared with cells treated with CP@HP-T, CDP@HP and CDP@HP-T at pH 7.4 (Fig. 5C), the cells’ death after US increased remarkably when they were treated at pH 6.5 (Fig. 5F), because pH-responsive nanoprobe accelerated drug release under the slight acidic pH. Taken together, the probe presented enhanced lethal effect of combined chemotherapy and SDT on cancer cell by the characteristics of acid response and mitochondrial targeting.

19

Fig. 6. The changes of white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), platelet (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum alkaline phosphatase (ALP), blood urea nitrogen(BUN) and various organ indexes (A) and HE staining sections (B) of mice after three injections of CDP@HP-T for 5 and 30 d. n=5.

20

2.3. In Vivo Biocompatibility of CDP@HP-T. Biosafety of nanoplatform was one of the necessary conditions for its application in vivo, so this factor of CDP@HP-T probe was evaluated in vivo. Healthy female Balb/C mice were randomly divided into three groups (n=5). Two groups were intravenously injected with CDP@HP-T (10 mg·kg-1 Ce6) every 3 d for three times, and observed for 5 and 30 d respectively. The other group was injected with the same amount of saline as control. At the end of the experiment, the blood biochemical indexes and organ indexes of mice were detected, and the organs were observed by HE staining. The result showed that the number of white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB) and platelet (PLT) in mice were not evidently different from those in control group for a short time (5 d) and a long time (30 d) after multiple injections of probe (Fig. 6A a-d), indicating that the probe had excellent hemocompatibility. Concomitantly, the damage of probe to liver and kidneys of mice was also evaluated. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were used as indicators of liver function, and blood urea nitrogen (BUN) and creatinine (CREA) were used as indicators of renal function. Compared with control group, there was no significant difference in ALT, AST, BUN and CREA whether for a short time or a long time (Fig. 6A e-h), testifying that the probe did not cause damage to liver and kidneys. Organ index as the sensitive indicator to organ was assessed. After multiple injections of probe for 5 and 30 d, there was no significant difference in organ index of heart, liver, spleen and lungs compared with control group (Fig. 6A i-l). HE staining result also revealed that the structure of heart, liver, spleen, lungs, kidneys and small intestine remained intact and without obvious damage and pathological change, which was consistent with control group (Fig. 6B). Moreover, HE and Nissl staining were used to observe the damage of probes on mice brain (Fig. S9). HE staining result revealed that the structure of mice

21

brains remained intact without obvious damage and pathological change, which was consistent with control group. And according to the results of Nissl staining, the number and morphology of Nissl bodies were consistent with those of the control group, indicating that the CDP@HP-T did not cause damage to the nerve cells of mice. To summarize, CDP@HP-T had excellent biocompatibility and could be used for in vivo experiment.

Fig. 7. Fluorescence imaging of 4T1 tumor-bearing mice, various organs and tumor at different time points after intravenous injection of CDP@HP-T (A); distribution of probe in major organs at different time points (B); pharmacokinetics of probe (C). n=3; **: p<0.01, ***: p<0.001.

22

2.4. In Vivo Imaging, Biodistribution and Pharmacokinetics. Next, the enrichment of CDP@HP-T in tumor and its distribution in various organs were further examined. To optimize the treatment time of mice, CDP@HP-T was injected into mice through tail vein, and then its enrichment in tumor site was monitored by fluorescence imaging. According to the fluorescence intensity increasing with the probe concentration (Fig. S10), it was found Ce6 fluorescence signal appeared at tumor site after 1 h and reached to a peak level at 3 h post injection (Fig. 7A), indicating efficient tumor uptake of CDP@HP-T. Moreover, fluorescence signal was also observed in the brain of mice. That was due to the plasma membrane of blood-brain barrier endothelial cell contains many negatively charged areas; consequently, positively charged substance could bind to these locations through electrostatic interaction and be readily internalized, and then exocytosed at the abluminal surface [36]. Then it was conjectured the modification of TPP on the surface of CDP@HP-T resulted in its enrichment in the brain of mice [37]. A similar phenomenon was also found in previous report, while TPP was also modified on the probe surface [11]. Furthermore, dopamine is a major component of naturally occurring melanin that is widely distributed in the human body, especially in brain as neurotransmitter. Then this might make dopamine based nanoparticle easier to get into the brain. Similar enrichments in the brains have also been observed in other dopamine based nanoparticles [18, 38-40]. After 5 min, 1, 3, 6, 12 and 24 h post injection, the main organs and tumor of mice were taken out for in vitro fluorescence imaging. It could be speculated that most probe was distributed in liver, spleen, lungs and kidneys after injection (Fig. 7B). The fluorescence intensities of these organs at 5 min were significantly higher than those at 12 h (p<0.001), demonstrating that the probe was gradually excluded from the mice with time. However, the fluorescence of tumor increased gradually and reached to the maximum at 3 h, which

23

was significant difference with other time points (p<0.01). The pharmacokinetic study of probe showed that the fluorescence intensity of blood in probe group was significantly higher than that of blank blood before 12 h (p<0.001). However, the fluorescence intensity of blood in probe group was gradually decreases with time, and there was no significant difference in the fluorescence intensity between the blood in probe group and the blank blood after 48 h, indicating that most of the probe was excluded from the mice after 48 h (Fig. 7C). And its half-life (t1/2) in blood was 3.49±0.87 h, which demonstrated the easy elimination of the probe from body. The above result confirmed that CDP@HP-T on one hand could effectively enrich in tumor site, providing a guarantee for the combined treatment of tumor, on the other hand could also be gradually metabolized out of body, showing that it had outstanding biological safety. 2.5. In Vivo combined chemo-SDT treatment with CDP@HP-T. As we know, compared with normal tissue, tumor site contains higher content of H2O2, which is expected to be decomposed by a certain catalytic reaction to up-regulate the oxygen level in TME [41]. To investigate the ability of probe to alleviate hypoxia in tumor, 4T1 tumor-bearing mice were intravenously injected with different probes and irradiated by US at 3 and 6 h. The tumors were stripped and stained with hypoxia marker pimonidazole antibody (green), anti-CD31 antibody (red) and nuclear marker 4',6-diamidyl-2-phenylindole (DAPI, blue) for immunofluorescence imaging after 24 h post injection. It was found that the mice injected with saline or Pt-free CD@HP-T probe showed bright green fluorescence at tumor sites after US except region nearby the blood vessel, just as control group (Fig. 8A). However, the green fluorescence of tumors from mice treated with CDP@HP-T was distinctly reduced after US, this was because CDP@HP-T could continuously decompose H2O2 to oxygen in tumor site, which improved the oxygen content in TME; however, in

24

other experimental groups, the tumor site could only rely on blood vessel transport to bring oxygen. Therefore, CDP@HP-T could effectively alleviate hypoxia in TME.

Fig. 8. Immunofluorescence staining micrograph of tumor slices collected from different treatment groups (A); body weight change (B), tumor volume change (C) and Kaplan-Meier survival plot (D) of tumor-bearing mice with various treatments; TUNEL and HE (E) staining of tumor after various treatments; n=5; **: p<0.01, ***: p<0.001.

25

Inspired by the above-mentioned result, the in vivo therapeutic effect of CDP@HP-T on tumor was studied. In experiment, 4T1 tumor-bearing mice were randomly divided into eight groups (n=6) and treated with different methods: (I) Saline, (II) Saline+US, (III) DOX, (IV) CDP@HP-T, (V) DP@HP-T+US, (VI) CP@HP-T+US, (VII) CDP@HP+US, (VIII)DC@HP-T+US and (IX) CDP@HP-T+US. The body-weight and tumor volume were measured every other day during experiment. The result showed that the body-weight of mouse in each group changed in the same way during 28 d, as that in control group (Fig. 8B), indicating probe and treatment did not cause harm to mice. The data of tumor volume showed that US alone could not inhibit the growth of tumor. After single chemotherapy (DOX, CDP@HP-T and DP@HP-T+US), the growth of tumor was pronouncedly inhibited, but it was still relatively rapid (Fig. 8C), indicating that the effect of single chemotherapy on tumor was limited. In Pt-assisted SDT group (CP@HP-T+US), Pt-free combined treatment group (CD@HP-T+US) and TPP-free combined treatment group (CDP@HP+US), the growth rates of tumor were further reduced and the inhibition effects were more obvious than those of single chemotherapy groups (p<0.01). Unfortunately, these three groups still could not completely inhibit the growth of tumor. Of particular note, only the Pt&TPP-assisted combined treatment group (CDP@HP-T+US) was able to eradicate the cancer (Fig. S11 and S12A), which was significantly different from other groups (p<0.01). This might be due to the following reasons. Firstly, CP@HP-T could achieve enhanced SDT after embedment of Pt and modification of mitochondrial targeted molecule TPP, but single SDT method could not effectively eradicate tumor. Secondly, in CD@HP-T+US group, because Pt who could decompose the H2O2 in TME to O2 was absent, SDT was limited by hypoxic environment, therefore, although combined with chemotherapy, the therapeutic effect was not good enough. Lastly, in CDP@HP+US group, the probe lacked the

26

mitochondrial targeted molecule TPP, so it could not directly damage the mitochondria, and then insufficient therapeutic effect was obtained. Concomitantly, the survival rate of mice in CDP@HP-T+US group was 100 % after 60 d treatment (Fig. 8D), while the mice in CP@HP-T+US, CDP@HP+US and CD@HP-T+US groups died in varying degree. The mice in control group and the single chemotherapy groups all died after 25 d and 30 d, respectively. And there was no obvious tumor mass in the original tumor-growing site of surviving mice (Fig. S13). HE staining for the organs of surviving mice were further performed. The results showed that the organs of mice were intact without pathological changes and metastasis (Fig. S14). Therefore, the above result demonstrated that CDP@HP-T+US group could effectively remove tumor, not only because it could achieve mitochondrial targeting and use Pt to enhance the effect of SDT, but also it could use weak acidic TME to promote drug release to obtain enhanced combination therapy of chemotherapy and SDT. TUNEL staining was further used to detect the damage of tumor in mice under different treatments after 24 h. The result showed that there was no obvious damage to tumor treated with US alone , but a small amount of apoptosis was observed in tumor treated with DOX, CDP@HP-T and DP@HP-T+US chemotherapy groups (Fig. 8E and S12B); however, in CP@HP-T+US, CDP@HP+US, CD@HP-T+US and CDP@HP-T+US groups, a large number of apoptotic cells were found, and the most obvious in the last group, which was consistent with the result of HE staining, demonstrating the smart ROS-augment nanoplatform could effectively improve the therapeutic efficacy of tumor. 3. Conclusion In this paper, a Pt-embedded hollow polydopamine nanoparticle co-loaded with DOX and Ce6

27

as smart pH-responsive and mitochondrial-targeted nanoplatform was developed to realize enhanced combination therapy of chemotherapy and SDT for cancer. The ultimate goal of our research is to promote the application of this ROS-based method in clinical treatment. Principally, biocompatibility and biodegradation are vital factor in determining the suitability of material for application in clinical applications. In our manuscript, the toxicity, distribution and pharmacokinetic of CDP@HP-T in mice and biodegradation of probe were evaluated. The hemolysis assay and in vivo toxicity test of probe showed that the probe had excellent biocompatibility and did not cause toxicity to mice. The biodegradability and pharmacokinetic experiment of probe demonstrated that it could be slowly degraded in vivo and excreted from the body, which provided a possibility for further application. All these experimental results confirmed that our probe had high biosafety. Furthermore, as a major component of naturally occurring melanin that is widely distributed in the human body, polydopamine show excellent biocompatibility and can significantly decrease the occurrence of serious adverse effects caused by the administration of foreign substance. After that, the therapeutic effect of our probe was evaluated. In vitro and in vivo experiments demonstrated that the probe could release drug after biodegradation in weakly acidic tumor microenvironment (TME) and/or lysosomal compartment, effectively relieve the hypoxic state of the tumor and carry drug to target mitochondria by TPP, resulting in a remarkable enhanced therapeutic effect of combined chemotherapy and SDT to eliminate tumor. Our nanoplatform will be beneficial to the further development of ROS-based fundamental researches and clinical applications. 4. Experimental Section Materials. Zinc nitrate hexahydrate (Zn(NO3)2·6H2O), 1,2-dimethylimidazole (2-MIL), (3-carboxypropyl)triphenylphosphonium bromide (TPP), 1,3-diphenyl isobenzofuran (DPBF),

28

2,2,6,6-tetramethylpiperidine (TEMP) and polyvinyl pyrrolidone (PVP) were purchased from Aladdin.

N-Hydroxysuccinimide

hydrochloride

(EDC),

(NHS),

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

chloroplatinic

acid

hydrate

(H2PtCl6·6H2O)

and

2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich. chlorine e6 (Ce6) was purchased from Frontier Scientifc Inc. Hoechst 33342 and Mito-Tracker Green were purchased from Beyotime Biotechnology. SH-PEG2000-NH2 was purchased from Xi'an Ruixi Biotechnology Co., Ltd. Hypoxyprobe-1 Plus Kit was purchased from Hypoxyprobe Inc. All the reagents used in the experiment were all of analytical grade and used directly without treatment. Characterizations. The absorption spectrum was performed by UV-2550 Ultraviolet-visible Spectrophotometer (Shimadzu, Japan). The size of probe was measured by ZS90 Zeta Sizer (Malvern, UK). Probe morphology was obtained on HT7700 transmission electron microscopy system (Hitachi, Japan). The probe material was analyzed by Empyrean XRD (PANalytical B.V., NED). WED-100 ultrasonic treatment apparatus (Welld, China) was used as the ultrasonic trigger source. The electron spin resonance spectrometer (Bruker EMXmicro-6/1, Germany) was used to detect the 1O2 generation. The in vivo fluorescence imaging system was established by our laboratory. Synthesis of P@ZIF-8. Firstly, PVP modified Pt nanoparticle was synthesized [42]. PVP (500 mg), methanol (150 mL) and chloroplatinic acid hydrate (6 mM, 20 mL) were mixed and stirred in a round bottom flask for 24 h under air. Then methanol was removed by rotating evaporation and Pt nanoparticle was precipitated with acetone (6000 rmp, 5 min). The sample was cleaned with chloroform and hexane to remove excess free PVP. Secondly, the zinc nitrate hexahydrate (25 mM) and 2-methyl imidazole (25 mM) methanol solution (40 mL) was rapidly stirred for 20 min and then adding PVP modified Pt nanoparticle (1 mL). After continuous stirring for 4 h, the sample washed 3

29

times with methanol. Eventually, P@ZIF-8 was obtained. Synthesis of SH-PEG2000-TPP. The TPP (20 mg), EDC (76.68 mg) and NHS (15.34 mg) were mixed in MES solution (5 mL) and activated for 30 min. Then pH was adjusted to 9 by triethylamine, and SH-PEG2000-NH2 (20 mg) was added. After continuous stirring for 12 h, SH-PEG2000-TPP was dialyzed with ultrapure water for 2 d and freeze-dried. Synthesis of CDP@HP-T. Firstly, hollow polydopamine embedded with platinum nanoparticle (P@HP) was synthesized. The P@ZIF-8 (4 mL), methanol (2.5 mL) and dopamine (2.5 mL, 20 mM) were mixed and refluxed at 60 °C. After 6 h, the sample (P@HP) was washed with methanol and ultra-pure water to remove free dopamine and 2-methylimidazole. Secondly, the P@HP (5 mg) and SH-PEG2000-TPP (5 mg) were mixed in PBS (5 mL) and stirred for 2 h, then DOX and Ce6 (dissolved in DMSO) were added to the mixed solution and stirred for another 12 h. The mixed solution was washed with ultra-pure water (10000 rmp, 10 min) to remove excess DOX and Ce6, and CDP@HP-T was obtained. The CDP@HP without targeting TPP was prepared by the same procedure using SH-PEG2000. Hemolysis Assay. The hemocompatibility of CDP@HP-T was studied by hemolysis reaction. In order to obtain red blood cell, fresh blood was washed repeatedly with PBS (3500 rpm, 5 min). CDP@HP-T with different concentrations was constant volume in 0.8 mL PBS and then adding 0.2 mL RBC solution. PBS and deionized water were negative and positive controls respectively, 3 parallel samples in each group. After incubation at 37 °C for 8 h, the mixture was centrifuged. ELX808IU microplate reader (Biotek, USA) was used to measure the absorption of supernatant at 540 nm. Hemolysis rate (%)=(ODtest-ODnegative control)/(ODpositive control-ODnegative control)×100 %. The drug loading and drug release of CDP@HP-T. To determine the drug loading of

30

CDP@HP-T, the P@HP (5 mg) and SH-PEG2000-TPP (5 mg) were mixed with different concentrations of DOX and Ce6 (dissolved in DMSO). After 12 h, the probe was washed with and ultra-pure water and supernatant was collected to detect its absorption value at 495 nm (DOX) and 660 nm (Ce6), respectively. The encapsulation efficiency of DOX/Ce6 in probe was calculated: (Input DOX/Ce6 quality-Quality of DOX/Ce6 in supernatant)/Input DOX/Ce6 quality×100 %。 To measure the release of DOX/Ce6 from probe at different pH, CDP@HP-T (0.5 mg) was dispersed in PBS at pH 7.4, 6.5 and 5.5 (5 mL) respectively, keeping in vapour-bathing table concentrator at 37 °C for 8 h. The solution was collected and centrifuged (10000 rpm, 10 min) at different time points, and the absorbance value of DOX/Ce6 in supernatant was measured at 495 nm (DOX) and 660 nm (Ce6), and then the supernatant was remixed with precipitate and put back to original solution. Three parallel samples were set for each group. Reactive Oxygen Species Production. The electron spin resonance spectrometer was used to detect the 1O2 generation by US-activated CDP@HP-T. TEMP, a 1O2 entrapment agent, was added to detect the ESR of CDP@HP-T, CDP@HP-T+US and CDP@HP-T+H2O2 (200 µΜ)+US groups (1.0 W·cm-2, 3 min). The yield of ROS produced by CDP@HP-T was also quantitatively analyzed by DPBF. The absorption value of DPBF and CDP@HP-T mixed solution at 410 nm was detected every 1 min. DPBF+H2O2 (200 µΜ) and DPBF+CDP@HP-T groups were used as controls. The remaining of DPBF=(Initial mixture absorption value-Absorption value after US)/Initial mixture absorption value×100 %. Catalytic decomposition of H2O2 by CDP@HP-T. CDP@HP-T was incubated with various concentrations of H2O2 deoxygenated by Ar (200 µΜ, 1 mM and 10 mM). The oxygen evolution over time of the above samples was recorded through a portable dissolved oxygen meter (Ray

31

magnetic, JPB-607A, China). To detect intracellular oxygen production, 4T1 cell was seeded in 6-well plate and cultured overnight. The medium containing P@HP-T or HP-T was added to the 6-well plate respectively, and the cell was incubated for 4 h. Then [Ru(dpp)3]Cl2 was added to each well as detection reagent for intracellular oxygen generation. After 30 min, the cell was washed with PBS for three times and observed by fluorescence microscopy. Hypoxic cell was obtained by adding deferoxamine in the medium and sealing with paroline. In vitro confocal fluorescence imaging. 4T1 cell was seeded in confocal dish and cultured overnight. The medium containing CP@HP-T or CP@HP was added to confocal dish respectively, and the cell was incubated for 4 h. Next, the cell was washed with PBS for three times, and stained with Hoechst 33342 (10 min) and Mito-Tracker Green (30 min). Finally, the cell was observed by laser confocal microscopy (Hoechst 33342, Excition: 405 nm, Mito-Tracker Green, Excition: 488 nm and Ce6, Excition: 559 nm). 4T1 cell was seeded in confocal dish and cultured overnight for pH-triggered drug release. Then cell was cultured with DP@HP-T under pH 7.4 or 6.5 medium, respectively. After 2 h, the cell was washed with PBS for three times and stained with Hoechst 33342 (10 min), then observed and photographed with laser confocal microscopy. In vitro ROS detected. ROS in cell was detected by DCFH-DA. Briefly, 4T1 cell was seeded in 6-well plate and cultured overnight. C@HP-T and CP@HP-T were used to incubate cell in dark for 4 h, then removing the medium and washing 3 times with PBS. Cell was further incubated with DCFH-DA for 30 min, and fluorescence imaging of cell was performed by fluorescence microscopy immediately after US irradiation (1.0 W·cm-2, 3 min).

32

In vitro Cytotoxicity Assay. The cytotoxicity against 3T3 and 4T1 cells was measured by MTT assay, the cells were cultured in 96-well plate and incubated overnight. Thereafter, the gradient concentrations of P@HP-T (10, 25, 50, 100, 150 and 200 µg·mL-1) were added into each well and incubated for 24 h, five parallel samples were set for each group. The standard thiazolyl tetrazolium test was applied to measure the cell viability. In vitro Synergistic Therapeutic Effect. The 4T1 cell was cultured in 96-well plate and incubated overnight. The medium was removed and 200 µL culture media containing different concentrations of CP@HP-T, CDP@HP and CDP@HP-T were added, respectively. After incubation for 4 h, the excess probe was removed with PBS, and then the cell was subjected to US. The survival rate of cell was calculated after continuous culture for 20 h. For the pH-responsive synergistic therapeutic effect, 4T1 cell was cultured in media (pH 6.5) containing CP@HP-T, CDP@HP and CDP@HP-T (2 µg·mL-1 Ce6) for 4 h. To observe the killing effect, Calcein-AM and PI staining were performed. The 4T1 cell was incubated in 6-well plate overnight. After the medium was removed, 1 mL CP@HP-T, CDP@HP and CDP@HP-T (2 µg·mL-1 Ce6) was added to the medium respectively for further incubation for 4 h. Then the cell was washed with PBS, and subjected to US (1.0 W·cm-2, 3 min) and stained by Calcein-AM and PI. The fluorescence imaging of cell was performed by fluorescence microscopy. In Vivo Safety Evaluation. The female BALB/c mice were divided into three groups randomly, two were injected with CDP@HP-T (10 mg·Kg-1 Ce6 and 5 mg·Kg-1 DOX) via tail vein every 3 d for three times. The other group was injected with the same amount of saline as control. The mice were sacrificed and blood was collected after 5 and 30 d, respectively. Blood biochemical analysis was performed, major organs (heart, liver, spleen, lungs, kidneys and small intestine) were weighed and

33

collected for HE staining. The same method was used to study the toxicity of the probe to mouse brain. The mice were brain was collected for HE and Nissl staining after 15 d. In vivo Fluorescence Imaging, Biodistribution and Pharmacokinetic Evaluation. The 4T1 cells (106) were subcutaneously inoculated in BALB/c nude mice (female, 4 weeks) to establish tumor model. After the tumor reached to 80~100 mm3, CDP@HP-T (10 mg·Kg-1 Ce6) was intravenously injected into tumor-bearing mice. Fluorescence imaging was performed in vivo at different time points (5 min, 1, 3, 6, 12 and 24 h), three five parallel samples were set for each group. To the biodistribution and pharmacokinetic evaluation of CDP@HP-T, 4T1 tumor-bearing mice (BALB/c) were injected with CDP@HP-T probe via the tail vein. After being sacrificed at different time points, each organ and tumor were collected. Kunming mice (female, SPF) were intravenously injected with CDP@HP-T, equal amounts of blood were collected at different time points (5 min, 30 min, 1, 3, 6, 12 and 24 h). The fluorescence imaging system was used to image each organ, tumor and blood. Three parallel samples were set for each group. In Vivo Synergistic Therapeutic Effect Evaluation. When the tumor reached to 80~100 mm3, 4T1 tumor-bearing mice were divided into eight groups randomly, six parallel samples were set for each group: I) Saline, (II) Saline+US, (III) DOX (5 mg·Kg-1 DOX), (IV) CDP@HP-T, (V) DP@HP-T+US, (VI) CP@HP-T+US, (VII) CDP@HP+US, (VIII)DC@HP-T+US and (IX) CDP@HP-T+US. After the different probes (10 mg·Kg-1 Ce6 and 5 mg·Kg-1 DOX) were intravenously injected into mice, US (1.5 W·cm-2, 5 min) was used to irradiate the experimental groups at 3 and 6 h. After 24 h of treatment, the solid tumor was dissected from one mouse in each group for HE and TUNEL staining. The solid tumor was also used for hypoxic immunohistochemical and anti-CD31 antibody immunohistochemical staining in (I), (II), (VII) and (VIII) group. The

34

pimonidazole (60 mg·kg-1) (Hypoxyprobe-1 Plus Kit, Hypoxyprobe Inc.) was intraperitoneal injected before 1.5 h surgical resection of every tumor. Body weight and tumor volume were recorded every other day. All animal experiments were approved by the Animal Experimental Ethics Committee of Huazhong University of Science. Data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. Acknowledgements

This work was supported by the National Key Research and Development Program of China (2017YFA0700402), the National Natural Science Foundation of China (Grant No. 81771878, 81971658, 91959109), and the Fundamental Research Funds for the Central Universities (Hust: 2016YXMS253, 2017KFXKJC002, 2018KFYXKJC048). We also thank the Analytical and Testing Center (HUST), the Research Core Facilities for Life Science (HUST) and the Center for Nanoscale Characterization & Devices (CNCD) at WNLO of HUST for the help of measurement. Prof. M.-Z. Zhang also thanked the support of “Young Talent Support Plan” of Xi’an Jiaotong University (YX6J001) and the Fundamental Research Funds for the Central Universities (xzy012019070). Appendix A. Supplementary data: Supplementary data to this article can be found online at:

References [1] B. Yang, Y. Chen, J. Shi. Reactive Oxygen Species (ROS)-Based Nanomedicine. Chem. Rev. 2019, 119, 4881-4985. [2] D. Trachootham, J. Alexandre, P. Huang. Targeting Cancer Cells by ROS-Mediated Mechanisms: 35

A Radical Therapeutic Approach? Nat. Rev. Drug Discovery 2009, 8, 579-591. [3] B. C. Dickinson, C. J. Chang. Chemistry and Biology of Reactive Oxygen Species in Signaling or Stress Responses. Nat. Chem. Biol. 2011, 7, 504-510. [4] J. Mou, T. Lin, F. Huang, H. Chen, J. Shi. Black Titania-based Theranostic Nanoplatform for Single NIR Laser Induced Dual-modal Imaging-guided PTT/PDT. Biomaterials 2016, 84, 13-24. [5] S. Wang, Z. Wang, G. Yu, Z. Zhou, O. Jacobson, et al. Tumor-Specific Drug Release and Reactive Oxygen Species Generation for Cancer Chemo/Chemodynamic Combination Therapy. Adv. Sci. 2019, 6, 1801986. [6] C. C. Winterbourn. Reconciling the Chemistry and Biology of Reactive Oxygen Species. Nat. Chem. Biol. 2008, 4, 278-286. [7] N. C. Denko. Hypoxia, HiF1 and Glucose Metabolism in the Solid Tumour. Nat. Rev. Cancer 2008, 8, 705-713. [8] F. Li, Y. Du, J. Liu, H. Sun, J. Wang, et al. Responsive Assembly of Upconversion Nanoparticles for pH-Activated and Near-Infrared-Triggered Photodynamic Therapy of Deep Tumors. Adv. Mater. 2018, 30, 1802808. [9] L. Song, P. P. Li, W. Yang, X. H. Lin, H. Liang, et al. Low-Dose X-Ray Activation of W(VI)-Doped Persistent Luminescence Nanoparticles for Deep-Tissue Photodynamic Therapy. Adv. Funct. Mater. 2018, 28, 1707496. [10] K. Cheng, X. Q. Yang, X. S. Zhang, J. Chen, J. An, et al. High-Security Nanocluster for Switching Photodynamic Combining Photothermal and Acid-Induced Drug Compliance Therapy Guided by Multimodal Active-Targeting Imaging. Adv. Funct. Mater. 2018, 28, 1803118. [11] G. Yang, L. Xu, J. Xu, R. Zhang, G. Song, et al. Smart Nanoreactors for pH-Responsive Tumor Homing, Mitochondria-Targeting, and Enhanced Photodynamic Immunotherapy of Cancer. Nano Lett. 2018, 18, 2475-2484. [12] N. Gong, X. Ma, X. Ye, Q. Zhou, X. Chen, et al. Carbon-dot-supported Atomically Dispersed Gold as a Mitochondrial Oxidative Stress Amplifier for Cancer Treatment. Nat. Nanotechnol. 2019, 14, 379-387. [13] K. Han, Q. Lei, S. B. Wang, J. J. Hu, W. X. Qiu, et al. Dual-Stage-Light-Guided Tumor Inhibition by Mitochondria Targeted Photodynamic Therapy. Adv. Funct. Mater. 2015, 25, 2961-2971. 36

[14] Y. Song, Q. Shi, C. Zhu, Y. Luo, Q. Lu, et al. Mitochondrial-targeted Multifunctional Mesoporous Au@Pt Nanoparticles for Dual-mode Photodynamic and Photothermal Therapy of Cancers. Nanoscale 2017, 9, 15813-15824. [15] J. R. Friedman, J. Nunnari. Mitochondrial Form and Function. Nature 2014, 505, 335-343. [16] H. Chen, J. Tian, W. He, Z. Guo. H2O2-Activatable and O2-Evolving Nanoparticles for Highly Efficient and Selective Photodynamic Therapy against Hypoxic Tumor Cells. J. Am. Chem. Soc. 2015, 137, 1539-1547. [17] P. Prasad, C. R. Gordijo, A. Z. Abbasi, A. Maeda, A. Ip, et al. Multifunctional Albumin-MnO2 Nanoparticles Modulate Solid Tumor Microenvironment by Attenuating Hypoxia, Acidosis, Vascular Endothelial Growth Factor and Enhance Radiation Response. ACS Nano 2014, 8, 3202-3212. [18] X. S. Wang, J. Y. Zeng, M. K. Zhang, X. Zeng, X. Z. Zhang. A Versatile Pt-Based Core–Shell Nanoplatform as a Nanofactory for Enhanced Tumor Therapy. Adv. Funct. Mater. 2018, 28, 1801783. [19] J. Kim, H. R. Cho, H. Jeon, D. Kim, C. Song, et al. Continuous O2 Evolving MnFe2O4 Nanoparticle-Anchored Mesoporous Silica Nanoparticles for Efficient Photodynamic Therapy in Hypoxic Cancer. J. Am. Chem. Soc. 2017, 139, 10992-10995. [20] X. Qian, X. Han, Y. Chen. Insights into the Unique Functionality of Inorganic Micro/Nanoparticles for Versatile Ultrasound Theranostics. Biomaterials 2017, 142, 13-30. [21] J. You, G. Zhang, C. Li. Exceptionally High Payload of Doxorubicin in Hollow Gold Nanospheres for Near-infrared Light-triggered Drug Release. ACS Nano 2010, 4, 1033-1041. [22] Q. Liu, N. Xu, L. Liu, J. Li, Y. Zhang, et al. Dacarbazine-Loaded Hollow Mesoporous Silica Nanoparticles Grafted with Folic Acid for Enhancing Antimetastatic Melanoma Response. ACS Appl. Mater. Interfaces 2017, 9, 21673-21687. [23]

G.

Yang,

L.

Xu,

Y.

Chao,

J.

Xu,

X.

Sun,

et

al.

Hollow

MnO2

as

a

Tumor-Microenvironment-Responsive Biodegradable Nano-platform for Combination Therapy Favoring Antitumor Immune Responses. Nat. Commun. 2017, 8, 902. [24] Y. Liu, K. Ai, L. Lu. Polydopamine and Its Derivative Materials: Synthesis and Promising Applications in Energy, Environmental, and Biomedical Fields. Chem. Rev. 2014, 114, 5057-5115. 37

[25] J. Yan, L. Yang, M. F. Lin, J. Ma, X. Lu, et al. Polydopamine Spheres as Active Templates for Convenient Synthesis of Various Nanostructures. Small 2013, 9, 596-603. [26] S. Xiang, H. J. Qian, Y. Chen, K. Zhang, Y. Shi, et al. Chelation Competition Induced Polymerization (CCIP): A Binding Energy Based Strategy for Nonspherical Polymer Nanocontainers’ Fabrication. Chem. Mat. 2017, 29, 6536-6543. [27] X. C. Liu, G. C. Wang, R. P. Liang, et al. Environment-Friendly Facile Synthesis of Pt Nanoparticles Supported on Polydopamine Modified Carbon Materials. J. Mater. Chem. A 2013, 1, 3945-3953. [28] Z. W. Li, C. Wang, L. Cheng, H. Gong, S. G. Yin, et al. PEG-Functionalized Iron Oxide Nanoclusters Loaded with Chlorin e6 for Targeted, NIR Light Induced, Photodynamic Therapy. Biomaterials 2013, 34, 9160-9170. [29] S. Mura, J. Nicolas, P. Couvreur. Stimuli-responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 991-1003. [30] F. Bernsmann, V. Ball, F. Addiego, A. Ponche, M. Miche, et al. Dopamine-Melanin Film Deposition Depends on the Used Oxidant and Buffer Solution. Langmuir 2011, 27, 2819-2825 [31] Z. Dong, L. Z. Feng, Y. Hao, M. Chen, M. Gao, et al. Synthesis of Hollow Biomineralized CaCO3−Polydopamine Nanoparticles for Multimodal Imaging-Guided Cancer Photodynamic Therapy with Reduced Skin Photosensitivity. J. Am. Chem. Soc. 2018, 140, 2165-2178. [32] L. Ding, X. B. Zhu, Y. L. Wang, B. Y. Shi, X, Ling, et al. Intracellular Fate of Nanoparticles with Polydopamine Surface Engineering and a Novel Strategy for Exocytosis-Inhibiting, Lysosome Impairment-Based Cancer Therapy. Nano Lett. 2017, 17, 6790-6801. [33] W. Cheng, C. Y. Ling, L. Xu, G. Liu, N. S. Gao, et al. TPGS-Functionalized Polydopamine-Modified Mesoporous Silica as Drug Nanocarriers for Enhanced Lung Cancer Chemotherapy against Multidrug Resistance. Small 2017, 13, 1700623. [34] P. Huang, X. Qian, Y. Chen, L. Yu, H. Lin, et al. Metalloporphyrin-Encapsulated Biodegradable Nanosystems for Highly Efficient Magnetic Resonance Imaging-Guided Sonodynamic Cancer Therapy. J. Am. Chem. Soc. 2017, 139, 1275-1284. [35] N. Li, C. Huang, Y. Luan, A. Song, Y. Song, S. Garg. Active Targeting Co-delivery System Based on pH-sensitive Methoxy-poly(ethylene glycol)2K-poly(ε-caprolactone)4K-poly(glutamic acid)1K for Enhanced Cancer Therapy J. Colloid Interface Sci. 2016, 472, 90-98. 38

[36] M. H. Li, Z. Luo, Z. N. Xia, X. K. Shen, K. Y. Cai. Time-Sequenced Drug Delivery Approaches towards Effective Chemotherapeutic Treatment of Glioma. Mater. Horiz. 2017, 4, 977-996. [37] S. Ghosh, S. Sarkar, S. T. Choudhury, T. Ghosh, N. Das. Triphenyl Phosphonium Coated Nano-quercetin for Oral Delivery: Neuroprotective Effects in Attenuating Age Related Global Moderate Cerebral Ischemia Reperfusion Injury in Rats. Nanomed.-Nanotechnol. Biol. Med. 2017, 13, 2439-2450. [38] D. Wu, X. H. Duan, Q. Q. Guan, J. Liu, X. Yang, et al. Mesoporous Polydopamine Carrying Manganese Carbonyl Responds to Tumor Microenvironment for Multimodal Imaging-Guided Cancer Therapy. Adv. Funct. Mater. 2019, 29, 1900095. [39] L. H. Shao, R. R. Zhang, J. Q. Lu, C. Y. Zhao, X. Deng, et al. Mesoporous Silica Coated Polydopamine

Functionalized

Reduced

Graphene

Oxide

for

Synergistic

Targeted

Chemo-Photothermal Therapy. ACS Appl. Mater. Interfaces 2017, 9, 1226-1236. [40] X. Y. Zhong, K. Yang, Z. L. Dong, X. Yi, Y. Wang, et al. Polydopamine as a Biocompatible Multifunctional Nanocarrier for Combined Radioisotope Therapy and Chemotherapy of Cancer. Adv. Funct. Mater. 2015, 25, 7327-7336. [41] D. He, L. Hai, X. He, X. Yang, H. W. Li. Glutathione-Activatable and O2/Mn2+-Evolving Nanocomposite for Highly Efficient and Selective Photodynamic and Gene-Silencing Dual Therapy. Adv. Funct. Mater. 2017, 27, 1704089. [42] G. Lu, S. Li, Z. Guo, O. K. Farha, B. G. Hauser, et al. Imparting Functionality to A Metal-Organic Framework Material by Controlled Nanoparticle Encapsulation. Nat. Chem. 2012, 4, 310-316.

39

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: