Smart Nanoreactors for pH-Responsive Tumor ... - ACS Publications

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Smart nano-reactors for pH-responsive tumor homing, mitochondriatargeting, and enhanced photodynamic-immunotherapy of cancer Guangbao Yang, Ligeng Xu, Jun Xu, Rui Zhang, Guosheng Song, Yu Chao, Liangzhu Feng, Fengxuan Han, Ziliang Dong, Bin Li, and Zhuang Liu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b00040 • Publication Date (Web): 22 Mar 2018 Downloaded from http://pubs.acs.org on March 22, 2018

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Smart nano-reactors for pH-responsive tumor homing, mitochondria-targeting, and enhanced photodynamic-immunotherapy of cancer Guangbao Yang1, Ligeng Xu1, Jun Xu1, Rui Zhang1, Guosheng Song1, Yu Chao1, Liangzhu Feng1, Fengxuan Han2, Ziliang Dong1, Bin Li2*, Zhuang Liu1* 1

Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for

Carbon-Based Functional Materials & Devices, Soochow University, 199 Ren’ai Road, Suzhou, 215123, Jiangsu, China. 2

Orthopaedic Institute, Medical College, Soochow University, Suzhou, Jiangsu, China.

*E-mail: [email protected], [email protected]

Abstract Photodynamic therapy (PDT) is an oxygen-dependent light-triggered non-invasive therapeutic method showing many promising aspects in cancer treatment. For effective PDT, nanoscale carriers are often needed to realize tumor-targeted delivery of photosensitizers, which ideally should further target specific cell organelles that are most vulnerable to reactive oxygen species (ROS). Secondly, as oxygen is critical for PDT-induced cancer destruction, overcoming hypoxia existing in majority of solid tumors is important for optimizing PDT efficacy. Furthermore, as PDT is a localized treatment method, achieving systemic antitumor therapeutic outcomes with PDT would have tremendous clinical values. Aiming at addressing the above challenges, we design a unique type of enzyme-encapsulated, photosensitizer-loaded hollow silica nanoparticles with rationally designed surface engineering as smart nano-reactors. Such nanoparticles with pH responsive surface coating show enhanced retention responding the acidic tumor microenvironment, and are able to further target mitochondria, the cellular organelle most sensitive to ROS. Meanwhile, decomposition of

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tumor endogenous H2O2 triggered by those nano-reactors would lead to greatly relieved tumor hypoxia, further favoring in vivo PDT. Moreover, by combining our nanoparticle-based PDT with check-point-blockade therapy, systemic antitumor immune responses could be achieved to kill non-irradiated tumors 1-2 cm away, promising for metastasis inhibition.

Keywords: photodynamic therapy, nano-reactors, tumor hypoxia, mitochondria targeting, check-point-blockade therapy.

Photodynamic therapy (PDT) as a minimally invasive treatment strategy to kill tumors with high specificity has received tremendous attention over the past decade, in both preclinical studies and clinical practices.1-4 During PDT, photosensitizing (PS) molecules are activated after light irradiation with specific wavelength and then generate reactive oxygen species (ROSs), especially singlet oxygen (SO), to kill cancer cells.5-8 However, to optimize the overall therapeutic outcomes of PDT, there are a number of critical issues remain to be resolved. Firstly, since SO often has a short half-life (< 40 ns) and can be effective only within a rather low distance after generation (< 20 nm),9 it would be ideal to deliver PSs not only into tumor cells, but also to those critical subcellular organelles that are most sensitive to ROSs (e.g. mitochondria).10-12 Secondly, due to insufficient oxygen supply (hypoxia) within many types of solid tumors, the therapeutic efficacy of oxygen-dependent PDT would be largely limited.13, 14 Furthermore, conventional PDT as a light-triggered local treatment method is not able to attack non-irradiated tumors 1-2 cm away without direct light exposure, and thus not effective to control the tumor metastases.15 In order to overcome the above limitations of traditional PDT, various strategies have been


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explored in recent years.6, 16, 17 Numerous types of nanoscale delivery systems have been developed to realize tumor-targeted delivery of PS agents, relying on the enhanced permeability and retention (EPR) effect, recognizing tumor-specific receptors, or being responsive to the specific tumor microenvironment (e.g. slightly reduced pH).18-21 Beyond conventional tumor targeting, a number of smart PDT agents have also been designed to enable targeting of specific cell organelles such as mitochondria or nuclei that are vulnerable to ROS, so as to further optimize the efficacy of PDT.22-25 To overcome the PDT resistance for hypoxic tumors, many innovative strategies have been proposed to improve PDT-induced cancer destruction by increasing the tumor oxygenation.26-28 Furthermore, while early studies have discovered certain levels of immune responses after PDT, the combination of PDT with immunotherapeutic methods such as checkpoint blockade has recently attracted a great ideal of interests to realize abscopal effect for metastasis inhibition.29-31 However, surveying literature, there is no single system that is able to simultaneously achieve all of the above aims, to our best knowledge. Therefore, we design a multi-stage responsive smart nanoparticle system to enhance the therapeutic outcome of PDT by addressing the above-mentioned issues in conventional PDT. Hollow silica nanoparticles with catalase (CAT), a water-soluble H2O2-decomposing enzyme, encapsulated within their inner cavities, as well as chlorine e6 (Ce6), a PS agent, doped into the silica lattice structure, are fabricated via a simple one-pot reaction. The obtained nanoparticles are modified with, (3-carboxypropyl)triphenylphosphonium bromide (CTPP), a mitochondria targeting molecule, and further modified with an acidic pH responsive charge-convertible polymer by electrostatic interactions. The obtained nanoparticles have a number of unique features as an innovative smart PDT nano-agent. (1) Those pH-responsive nanoparticles once within the slightly acidic tumor


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microenvironment could be converted into positively charged particles with enhanced tumor cell internalization and tumor retention. (2) After pH-responsive deshielding of polymer coating, CTPP modified on the surface of naked nanoparticles would be exposed, enabling effective intracellular targeting of mitochondrial and thus further improved PDT cancer cell killing. (3) Catalase encapsulated within those nanoparticles would in the meanwhile act as nano-reactors to trigger decomposition of tumor endogenous H2O2, producing O2 to overcome tumor hypoxia and further enhance in vivo PDT efficacy. (4) After combination with check-point blockade immunotherapy with programmed death-ligand 1 (PD-L1) antibody, the enhanced PDT treatment could significantly promote infiltration of cytotoxic T lymphocytes (CTLs) into distant tumors and inhibit their growth, demonstrating a strong abscopal effect promising in metastasis inhibition.

The procedure for the fabrication of our smart nanoparticle system is illustrated in Figure 1a. Firstly, Chlorin e6 (Ce6) was covalently conjugated to (3-aminopropyl)triethoxysilane (APTES) and mixed with tetraethyl orthosilicate (TEOS) as the silica precursor. Interestingly, we uncovered that upon addition of proteins into the growth solution of silica nanoparticles, hollow silica nanoparticles with proteins encapsulated within the hollow cavities would be formed. Therefore, by adding catalase (CAT) into the growth solution containing both TEOS and APTES-Ce6, CAT-encapsulated, Ce6-doped hollow silica nanoparticles (CAT@S/Ce6) would be formed. Next, (3-carboxypropyl) triphenylphosphonium bromide (CTPP) was also covalently conjugated to APTES and then added into the solution of CAT@S/Ce6 to obtain CTPP modified CAT@S/Ce6 (CAT@S/Ce6-CTPP). At last, a pH-responsive anionic polymer, polyethylene glycol (PEG) / 2,3-dimethylmaleic anhydride (DMMA) co-grafted poly(allylamine hydrochloride) (PAH), which was abbreviated as DPEG, was


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prepared according to our previous protocol32 and used to modified positively charged CAT@S/Ce6-CTPP nanoparticles, obtaining CAT@S/Ce6-CTPP/DPEG as our final nanostructure. As the control, we used a pH-inert polymer, PEG / succinic anhydride (SA) co-grafted PAH (SPEG) to coat nanoparticles. The other controls including nanoparticles prepared by replacing CAT with bovine serum albumin (BSA) without the catalytic function (BSA@S/Ce6-CTPP/DPEG), as well as nanoparticles without CTPP modification (CAT@S/Ce6/DPEG), were also prepared following similar methods. Next, we carefully characterized the obtained nanoparticles. Under transmission electron microscope (TEM) imaging (Figure 1b), the obtained CAT@S/Ce6-CTPP/DPEG show well-defined hollow structure with quite uniform size at ~100 nm, and shell thickness at ~12 nm. Elemental mapping by the high-angle annular dark-field scanning TEM (HAADF-STEM) revealed the uniform distribution of Si, O and P elements, demonstrating successful CTPP modification on those nanoparticles (Figure 1c). Moreover, Ce6 characteristic absorbance peaks showed up in the UV-vis-NIR absorbance spectra of those nanoparticles, evidencing efficient Ce6 loading within such CAT@S/Ce6-CTPP/DPEG nanoparticles (Figure S1). Furthermore, PEG offered nanoparticles well dispersion in different solutions (Figure S2). In our previous work, we have developed pH-responsive charge-reversible upconversion nanoparticles (UCNPs) for in vivo PDT.32 Those nanoparticles under pH 6.8 would undergo rapid charge conversion from negative to positive, enabling enhanced cellular internalization of nanoparticles and increased PDT efficacy within the acidic tumor microenvironment. In this work, we used the same pH-sensitive polymer to coat our synthesized nanoparticles by electrostatic interactions. As shown Figure 1d, the zeta potential of CAT@S/Ce6-CTPP/DPEG increased from


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around -18 mV to +11 mV within 4 h at pH 6.8. While the zeta potential of those nanoparticles kept negative over 4 h at pH 7.4. As a control, CAT@S/Ce6-CTPP/SPEG nanoparticles coated with pH-inert polymers showed constant negative zeta potentials at either pH 6.8 or 7.4, suggesting the charge conversion of CAT@S/Ce6-CTPP/DPEG nanoparticles were indeed due to their surface polymer coating with pH responsive DPEG. Catalase is an enzyme to trigger the rapid decomposition of H2O2 into H2O and O2.33-36 To evaluate the catalytic activity of catalase loaded inside our hollow nanoparticles, we used a dissolved oxygen meter to measure oxygen concentrations in the H2O2 solution (500 µM) after adding CAT@S/Ce6-CTPP/DPEG or BSA@S/Ce6-CTPP/DPEG (Figure 1e). While rapid oxygen generation in the H2O2 solution was observed after CAT@S/Ce6-CTPP/DPEG was added, BSA@S/Ce6-CTPP/DPEG showed no catalytic activity to decompose H2O2. Moreover, CAT@S/Ce6-CTPP/DPEG exhibited obvious concentration-dependent H2O2 decomposition function (Figure 1f). We further measured the enzymatic stability of CAT@S/Ce6-CTPP/DPEG in the presence of proteases, which are widely found in physiological environment, by the Góth method.37 While free catalase completely lost its catalytic activity after incubation with protease K for 24 h, catalase loaded in CAT@S/Ce6-CTPP/DPEG was well protected and kept 70 % of its initial activity after the same treatment (Figure 1g), demonstrating the excellent enzyme activity protection effect of the silica shell. Next, we tested the light-triggered singlet oxygen (SO) generation efficiencies of Ce6-loaded nanoparticles. Free Ce6, BSA@S/Ce6-CTPP/DPEG and CAT@S/Ce6-CTPP/DPEG samples in the presence or absence of H2O2 were exposed to a 660-nm light. SO generation was tested by a probe of Singlet oxygen sensor green (SOSG) under light irradiation (Figure S3). Interestingly, the


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light-induced SO production by CAT@S/Ce6-CTPP/DPEG was significantly enhanced after addition of H2O2, which however had no noticeable effect to the light-induced SO generation by either free Ce6 or BSA@S/Ce6-CTPP/DPEG. Therefore, the additional oxygen generated by decomposition of H2O2 triggered by CAT@S/Ce6-CTPP/DPEG would be favorable for more efficient PDT. Next, we studied the efficacy of CAT@S/Ce6-CTPP/DPEG for in vitro PDT (Figure 2a). We firstly measured the cytotoxicity of CAT@S/Ce6-CTPP/DPEG without light irradiation (Figure 2b). After incubation for 24 h, the cell viability assay revealed no appreciable dark toxicity of CAT@S/Ce6-CTPP/DPEG to 4T1 murine breast cancer cells. Considering the hypoxic tumor microenvironment, we then tested the therapeutic efficacy of BSA@S/Ce6-CTPP/DPEG and CAT@S/Ce6-CTPP/DPEG under the oxygen-deficient nitrogen atmosphere (Figure 2c). After incubation with nanoparticles for 2 h, 4T1 cells were irradiated by 660 nm light for 1 h (5 mW cm-2) and then incubated for another 24 h within the standard atmosphere before the cell viability assay. The light-induced cell killing efficacy of CAT@S/Ce6-CTPP/DPEG was much higher than that of BSA@S/Ce6-CTPP/DPEG, owing to the additional oxygen supplied via decomposing endogenic H2O2

produced

by

tumor

cells

even

within

the

nitrogen

atmosphere.

Therefore,

CAT@S/Ce6-CTPP/DPEG could be used for efficient PDT treatment even in the hypoxic tumor microenvironment. The pH-responsive in vitro PDT with our nanoparticles were then studied. 4T1 cells were incubated with CAT@S/Ce6-CTPP/SPEG or CAT@S/Ce6-CTPP/DPEG at pH 7.4 or 6.8 for 4 h. As observed by confocal fluorescence imaging (Figure 2d), CAT@S/Ce6-CTPP/DPEG nanoparticles showed obviously increased cellular uptake under reduced pH at 6.8, as compared to that at pH 7.4. In contrast, the cellular uptake of CAT@S/Ce6-CTPP/SPEG was not notably affected by varying the


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incubation pH. For pH-responsive PDT experiments, 4T1 cells were incubated with CAT@S/Ce6-CTPP/SPEG or CAT@S/Ce6-CTPP/DPEG under different pH values (7.4 and 6.8) for 4 h. Thereafter, cells were conducted with PBS and then irradiated by 660-nm light (5 mW cm-2, 1 h). The cell viability assay was processed after incubation in cell medium for another 24 h (Figure 2e). Compared

to

cells

treated

with

CAT@S/Ce6-CTPP/SPEG

(pH

7.4

or

6.8)

or

CAT@S/Ce6-CTPP/DPEG at pH 7.4, the cell death after PDT increased remarkably when they were treated with CAT@S/Ce6-CTPP/DPEG at pH 6.8, owing to the enhanced cellular uptake of those pH-responsive nanoparticles under the slightly acidic pH. Therefore, CAT@S/Ce6-CTPP/DPEG nanoparticles with pH-responsive deshielding of surface PEGylation and charge-conversion properties could be employed as a smart PDT agent with enhanced cell uptake and killing efficacy under slightly reduced pH. Next, to further demonstrate whether the CAT@S/Ce6-CTPP/DPEG could target mitochondria of cancer cells, we compared the intracellular localization of nanoparticles with and without CTPP modification by confocal imaging to track Ce6 fluorescence signals. The Ce6 fluorescence within cells incubated with CAT@S/Ce6-CTPP/DPEG showed gradual overlap with the signals of MitoTracker by prolonging of the incubation time (Figure 2f), suggesting the specific intracellular targeting of mitochondria by those nanoparticles. In contrast, the Ce6 and MitoTracker fluorescence signals in cells incubated with CAT@S/Ce6/DPEG (without CTPP modification) showed less co-localization. The Pearson’s colocalization coefficient (Rr), which could be used to determine the co-localization of two types of fluorescence signals, was calculated to be 0.56 for CAT@S/Ce6-CTPP/DPEG and 0.13 for CAT@S/Ce6/DPEG, after 8 h incubation time for the incubated cells, further evidencing the improved mitochondria targeting of CTPP conjugated


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nanoparticles (Figure 2g). Previous studies have reported that many positively charged nanoparticles can escape from endosomes/lysomes after internalization and exhibit endosomal escape because of the “proton-sponge” effect.38-40 Therefore, CAT@S/Ce6-CTPP/DPEG nanoparticles under pH 6.8 would undergo rapid charge conversion from negative to positive, enabling induced endosomal escape of nanoparticles and further targeting to mitochondria. As the results of the mitochondria targeting ability of CAT@S/Ce6-CTPP/DPEG, significantly enhanced cancer cell destruction under PDT

was

observed

for

CAT@S/Ce6-CTPP/DPEG,

compared

to

that

achieved

with

CAT@S/Ce6/DPEG (Figure 2h). Those results indicated that CAT@S/Ce6-CTPP/DPEG could efficiently target mitochondria after being internalized by cancer cells, further enhancing the treatment efficacy of PDT. After comprehensive in vitro assays, we then evaluated in animal experiments. In vivo fluorescence imaging was studied to track in vivo behaviors of CAT@S/Ce6-CTPP/SPEG and CAT@S/Ce6-CTPP/DPEG (Ce6 = 5 mg kg-1) after intravenous (i.v.) injection into mice. Mice treated with CAT@S/Ce6-CTPP/DPEG showed strong fluorescence signals at the tumor site, indicating efficient tumor uptake of those charge-reversible nanoparticles (Figure 3a). In contrast, much

weaker fluorescence

was

detected

in

the

tumor

on

mice

i.v.

injected

with

CAT@S/Ce6-CTPP/SPEG (Figure 3b). Semiquantitative data illustrated that the tumor uptake of CAT@S/Ce6-CTPP/DPEG was 1.7-fold compared with that of CAT@S/Ce6-CTPP/SPEG (Figure 3c). Moreover, confocal fluorescence images of tumor slices showed that the Ce6 fluorescence in the CAT@S/Ce6-CTPP/DPEG

group

was significantly higher than that in

the

group

of

CAT@S/Ce6-CTPP/SPEG (Figure 3d), further confirming the improved tumor uptake of CAT@S/Ce6-CTPP/DPEG nanoparticles. Similar to many other previous reports on nanoparticles


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with pH-responsive charge conversion ability, it is thus speculated that CAT@S/Ce6-CTPP/DPEG once within the acidic tumor microenvironment would be converted into positively charged nanoparticles, which would exhibit greatly enhanced binding and uptake by tumor cells, favoring more efficient tumor retention. Additionally, quantitative biodistribution measurement by detecting Si levels in different organs using inductively-coupled plasma atomic-emission spectroscopy (ICP-AES) evidenced the reasonably long blood half-life and decent tumor uptake of CAT@S/Ce6-CTPP/DPEG nanoparticles after i.v. injection (Figure S4). Previous studies have demonstrated that the absorbance of hemoglobin at 750 nm and 850 nm could be employed for PA imaging to analyze the blood oxygenation status.41, 42 In our experiment, PBS, free catalase, BSA@S/Ce6-CTPP/DPEG and CAT@S/Ce6-CTPP/DPEG (CAT = 3.8 mg kg-1, Ce6 = 5 mg kg-1) were i.v. injected into 4T1 tumor model (Figure 3e). It was observed that the oxygenation levels in tumor area were significantly increased for mice treated with CAT@S/Ce6-CTPP/DPEG (Figure 3f). However, ineffective hypoxia level reduction for mice i.v. injected with free catalase or BSA@S/Ce6-CTPP/DPEG at the same respective doses. Furthermore, for immunofluorescence staining, pimonidazole hydrochloride as a hypoxyprobe was employed to test tumor hypoxia status after various treatments (Figure 3g). Consistent to PA imaging data, CAT@S/Ce6-CTPP/DPEG (CAT = 3.8 mg kg-1, Ce6 = 5 mg kg-1) could remarkably decreased the hypoxia-specific fluorescence signals (green) originated from anti-pimonidazole in exterior, intermediate and interior regions of a tumor, suggesting the greatly reduced hypoxia for the entire tumor in this group. Our results taken together evidenced that the tumor oxygenation could be greatly improved by i.v. injection of CAT@S/Ce6-CTPP/DPEG, owing to the decomposition of endogenous H2O2 in the tumor microenvironment triggered by catalase loaded inside those nanoparticles. Free


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catalase, on the other hand, was not that effective in promoting tumor oxygenation, likely owing to the limited tumor retention and relatively low stability against proteases of such free enzyme. To study the therapeutic efficacy of CAT@S/Ce6-CTPP/DPEG nanoparticles for 4T1 tumor model, the mice were i.v. injected with PBS (Group 1), free Ce6 (Group 2), CAT@S/Ce6/DPEG (Group 3), BSA@S/Ce6-CTPP/DPEG

(Group

4),

CAT@S/Ce6-CTPP/SPEG

(Group

5),

or

CAT@S/Ce6-CTPP/DPEG (Group 6). At 24 h post injection of various therapeutic agents (CAT = 3.8 mg kg-1, Ce6 = 5 mg kg-1), group 2, 3, 4, 5, 6 were irradiated by 660-nm light (5 mW cm-2, 1 h). In the following two weeks, the tumor volumes and body weights of mice were closely measured. As shown in Figure 4a, free Ce6 with 660-nm light irradiation (Group 2) was ineffective in tumor suppression, likely due to the limited tumor accumulation of free Ce6. The tumor growth was partially delayed by PDT with CAT@S/Ce6/DPEG (Group 3), BSA@S/Ce6-CTPP/DPEG (Group 4) and

CAT@S/Ce6-CTPP/SPEG

(Group

5).

Notably,

for

mice

after

PDT

with

CAT@S/Ce6-CTPP/DPEG (Group 6), the most significant inhibition of tumor growth was observed. The average tumor weights (Figure 4b) and corresponding photos of tumors (Figure 4c) from different groups both illustrated the best antitumor efficacy of PDT with CAT@S/Ce6-CTPP/DPEG. Therefore, the relief of tumor hypoxia by CAT, the mitochondria targeting by CTPP, as well as pH-responsive charge conversion ability offered by DPEG, are all important to enhance PDT therapeutic effect in our system. In addition, hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was conducted for tumor slices after various treatments. It was found that CAT@S/Ce6-CTPP/DPEG injection plus light irradiation revealed the highest level of cancer cells apoptosis compared to the other groups (Figure 4e), verifying our


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observed therapeutic effects based on tumor sizes. Moreover, no obvious variations of mouse body weights were observed in all groups following the process of treatment (Figure 4d), and no noticeable organ damage was observed after PDT with CAT@S/Ce6-CTPP/DPEG (Figure S5), both suggesting the excellent biocompatibility of those nanocarriers. In recent years, cancer immunotherapy has shown many exciting clinical results to treat different types of cancers.29, 43 However, their clinical responses are still limited because of the heterogeneity of tumors and complicated tumor microenvironment.44 Accumulating evidences by many different groups including ours have demonstrated that cancer immunotherapy combining with other treatment modalities would offer additional benefit in cancer treatment.26, 29 Therefore, in this study, we further combined CAT@S/Ce6-CTPP/DPEG-based PDT with programmed cell death 1 ligand (PD-L1) checkpoint blockade, which could effectively enhance the antitumor immune activity of CTLs by preventing their exhaustion. The approach reported here might provide an alternative strategy to eliminate primary tumors and kill spreading metastatic tumors. In our experiments, 4T1 tumor cells were inoculated on the left and right flanks of each mouse. The left and right tumors of mice were selected as primary tumors with PDT treatment, and distant tumors (1-2 cm away) without direct light exposure, respectively (Figure S6). Balb/c mice bearing 4T1 tumors were divided into four groups: Untreated (1), CAT@S/Ce6-CTPP/DPEG plus anti-PD-L1

(2),

CAT@S/Ce6-CTPP/DPEG

with

660-nm

light

irradiation

(3),

CAT@S/Ce6-CTPP/DPEG with 660-nm light irradiation and plus anti-PD-L1 (4). After 24 h i.v. injection of different therapeutic agents, the left tumors of mice from group 3 and group 4 were exposed to 660-nm light irradiation (5 mW cm-2, 1 h). At day 1, 3, 5, Anti-PD-L1 antibody was i.v. injected into mice in group 2 and group 4 at a dose of 750 µg/kg post light irradiation (Figure 5a).


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It was found that PDT plus anti-PD-L1 treatment (Group 4) could more efficiently inhibit the primary tumor progression than PDT treatment alone with CAT@S/Ce6-CTPP/DPEG nanoparticles (Group 3), while treatment with nanoparticles in the absence of light plus anti-PD-L1 (Group 2) showed no significant therapeutic efficacy at our used anti-PD-L1 dose (Figure 5b, c). Interestingly, distinguished from other treatments, only PDT plus anti-PD-L1 treatment induced robust CD8+ cytotoxic T lymphocytes (CTL) infiltration (over 4 folds than others) in the primary tumor (Figure 5d). Most strikingly, it was observed that only PDT plus anti-PD-L1 treatment could effectively inhibit the progression of non-irradiated distant tumors (1-2 cm away), whose growth was not affected in other groups (Figure 5e, f). Similar, significantly increased CTL infiltration was also found in the distant tumors for mice after the combination treatment, compared to that in other groups (Figure 5g). The robust production of interferon gamma (IFN-γ) in the serum samples of mice after PDT plus anti-PD-L1 treatment measured at day 7 post irradiation also proved the highly effective cellular immune responses induced by the combination treatment (Figure 5h). In addition, the body weights showed no apparent changes in mice from different treatment groups (Figure 5i). All the above results indicated that our CAT@S/Ce6-CTPP/DPEG-based PDT combining anti-PD-L1 blockade could synergistically induce highly effective antitumor immune responses to not only destruct tumors with direct PDT treatment, but also combat progression of tumors without direct light irradiation. As summarized in Figure 5j, CAT@S/Ce6-CTPP/DPEG nanoparticles could offer enhanced PDT performance owing to their pH-responsive tumor retention, mitochondria targeting, and tumor hypoxia relief functions. Furthermore, the local PDT treatment with CAT@S/Ce6-CTPP/DPEG could not only destroy cancer cells under light exposure, but also trigger systemic antitumor immune


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responses, such as by releasing tumor-associated antigens (TAAs) from damaged tumor cells after PDT treatment.26, 31 Then, those TAAs would be engulfed, processed and presented by professional antigen presenting cells like dendritic cells to activate CTLs, whose activity could be protected and enhanced by anti-PD-L1 checkpoint blockade. Finally, CTLs would migrate into non-irradiated distant tumors and mediate cellular immunity to specifically eliminate spreading tumor cells. Therefore, our study offers a new strategy for killing cancer cells which can hardly be directly irradiated by light during PDT treatment, so as to treat metastatic tumors.

In this work, we developed pH-responsive charge-convertible, mitochondria-targeting and catalase-encapsulated hollow silica nanoparticles with Ce6 doping as a smart nano-reactor for improved cancer PDT. Those nanoparticles within the acidic tumor microenvironment would show charge-conversion from negative to positive, favoring their cellular internalization and tumor retention. The subcellular organelle (mitochondria) targeting ability of those nanoparticles could enhance PDT-induced cancer cell killing, by generating singlet oxygen in the mitochondria that is highly vulnerable to ROS. With catalase encapsulated and delivered into tumors, the designed CAT@S/Ce6-CTPP/DPEG nanoparticles are also able to decompose tumor endogenous H2O2 and overcome the tumor hypoxia, greatly enhancing PDT treatment of solid tumors. Furthermore, such smart nanoplatform-based PDT shows great synergistic effects when combined with anti-PD-L1 checkpoint blockade to induce robust antitumor immunities, which can suppress the growth of tumors without direct light exposure. Such a strategy utilizing our newly designed smart nanoparticles are able to simultaneously address several limitations of conventional PDT mentioned above, and may be promising in treating both local tumors as well as distant tumor metastases.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental

details,

UV-vis-NIR

absorption

spectra,

Average

size

changes

of

CAT@S/Ce6-CTPP/DPEG incubated in different solutions, the production of singlet oxygen for different nanoparticles, blood circulation and biodistribution of CAT@S/Ce6-CTPP/DPEG, H&E stained images of major organs, photos to show the set up for a mouse under light irradiation. (PDF)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected], [email protected] ORCID Guangbao Yang: 0000-0001-6180-5310 Liangzhu Feng: 0000-0002-2712-050X Bin Li: 0000-0001-8516-9953 Zhuang Liu: 0000-0002-1629-1039 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT: This work was partially supported by the National Research Programs from Ministry of Science and Technology (MOST) of China (2016YFA0201200), the National


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Natural Science Foundation of China (51525203, 81403120, 31530024), Collaborative Innovation Center of Suzhou Nano Science and Technology, and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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Figure 1. Preparation and characterization of CAT@S/Ce6-CTPP/DPEG. (a) A scheme illustration for synthesis of CAT@S/Ce6-CTPP/DPEG nanoparticles. (b) TEM image of CAT@S/Ce6-CTPP/DPEG. Inset shows a magnified TEM image. (c) HAADF-STEM image and element mapping for CAT@S/Ce6-CTPP/DPEG nanoparticles. (d) Zeta potential changes of CAT@S/Ce6-CTPP/SPEG and CAT@S/Ce6-CTPP/DPEG in different pH values (6.8 and 7.4). (e) The oxygen production in H2O2 solutions after incubation with BSA@S/Ce6-CTPP/DPEG or CAT@S/Ce6-CTPP/DPEG. (f) The oxygen production in H2O2 solutions after incubation with different concentrations of CAT@S/Ce6-CTPP/DPEG. (g) The relative enzymatic activity changes of free catalase and CAT@S/Ce6-CTPP/DPEG after incubation with protease K.


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Figure 2. In vitro PDT experiments. (a) Schematic diagram of the enhanced PDT process with CAT@S/Ce6-CTPP/DPEG. (b) Relative viabilities of 4T1 cells treated with various concentrations of CAT@S/Ce6-CTPP/DPEG in the dark. (c) Relative viabilities of 4T1 cells conducted by BSA@S/Ce6-CTPP/DPEG or CAT@S/Ce6-CTPP/DPEG within the hypoxic atmosphere (1 % O2 and 5 % CO2 balanced with N2) for 2 h and then exposed to 660-nm light irradiation (5 mW cm-2, 1 h). (d) Confocal images of 4T1 cells after incubation with CAT@S/Ce6-CTPP/SPEG or CAT@S/Ce6-CTPP/DPEG at different pH values (6.8 and 7.4). (e) Relative viabilities of 4T1 cells after incubation with CAT@S/Ce6-CTPP/SPEG or CAT@S/Ce6-CTPP/DPEG at pH 6.8 or 7.4 for 4 h and then irradiated by the 660-nm light (5 mW cm-2 for 1 h). (f) Confocal images to show subcellular localization of CAT@S/Ce6/DPEG and CAT@S/Ce6-CTPP/DPEG with different


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incubation time. (g) The statistic of Pearson’s correlation coefficient (Rr) from (f). (h) Relative 4T1 cell viabilities with the treatment of CAT@S/Ce6/DPEG or CAT@S/Ce6-CTPP/DPEG for 12 h and then exposed to the 660-nm light irradiation (5 mW cm-2, 1 h).


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Figure 3. In vivo imaging and tumor oxygenation modulation. (a) In vivo fluorescence imaging of mice post injection of CAT@S/Ce6-CTPP/SPEG or CAT@S/Ce6-CTPP/DPEG. (b) Ex vivo fluorescence images of normal organs and tumors taken at 8 h after i.v. injection of these two types of nanoparticles. H, Lu, Ki, Sp, Li and Tu stand for heart, lung, kidney, spleen, liver and tumor, respectively. (c) Quantification of ex vivo fluorescence intensities of the major organs and tumors in (b). (d) Confocal images of tumor slices taken from mice as showed in (b). Blue and green represent DAPI (cell nuclei) and Ce6 fluorescence (nanoparticles), respectively. (e) PA imaging of 4T1


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tumor-bearing mice to test the tumor oxygenation status before and 24 h after i.v. injection of different nanoparticles. (f) Quantification of the oxyhemoglobin saturation in the tumor based on PA imaging data in (e). (g) Immunofluorescence staining micrographs of tumor slices collected from different treatment groups. The cell nuclei, hypoxia areas and blood vessels were stained by DAPI (blue), anti-pimonidazole antibody (green) and anti-CD 31 (red), respectively. P values were calculated based on Tukey’s post-test (***p < 0.001, **p < 0.01, or *p < 0.05).


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Figure 4. In vivo PDT treatment with CAT@S/Ce6-CTPP/DPEG. (a) Tumor growth curves after PDT treatment in 4T1 model. Error bars indicated the standard deviations of five mice per group. (b) The average tumor weights of different groups taken out from mice at the end of treatment. (c) A photograph of all tumors taken from various groups of mice at the end of study. (d) Body weights change of different groups over time. (e) H&E stained and TUNEL stained tumor slices taken at day 5 after different treatments. P values were calculated based on Tukey’s post-test (***p < 0.001, **p < 0.01, or *p < 0.05).


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Figure 5. The abscopal effect of PDT with CAT@S/Ce6-CTPP/DPEG in combination with checkpoint blockade immunotherapy. (a) Schematic illustration to show the experimental design of combing PDT with anti-PD-L1 therapy. (b-d) The tumor growth curves (b), average tumor weights at


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day 18 (c), and percentages of CTL infiltration at day 18 (d), for primary tumors (left) after various treatments indicated. (e-g) The tumor growth curves (e), average tumor weights at day 18 (f), and percentages of CTL infiltration at day 18 (g), for non-irradiated tumors (right) after various treatments indicated. Data are presented as means ± standard deviations (n = 5). (h) The IFN-γ levels in sera from mice detected at 7 days after various treatments. (i) Changes in body weight of mice during treatment. (j) A scheme indicating the mechanisms of combining PDT with anti-PD-L1 therapy. P values in (b), (c), (d), (e), (f), (g) and (h) were calculated by Tukey’s post-test (***p < 0.001, **p < 0.01, or *p < 0.05).


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TOC Figure:


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