Photodynamic and Combined

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Biological and Medical Applications of Materials and Interfaces

Temporally-Controlled Photothermal/Photodynamic and Combined Therapy for Overcoming Multidrug Resistance of Cancer by Polydopamine Nanoclustered Micelles Yuxin Xing, Tao Ding, Zhenqiang Wang, Liucan Wang, Haidi Guan, Jia Tang, Dong Mo, and Jixi Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 26, 2019

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ACS Applied Materials & Interfaces

Temporally-Controlled Photothermal/Photodynamic and Combined Therapy for Overcoming Multidrug Resistance of Cancer by Polydopamine Nanoclustered Micelles

Yuxin Xing, Tao Ding, Zhenqiang Wang, Liucan Wang, Haidi Guan, Jia Tang, Dong Mo, Jixi Zhang*

Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, No. 174 Shazheng Road, Chongqing 400044, China

*e-mail: [email protected]

KEYWORDS

ACS Paragon Plus Environment

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disintegratable nanoclusters, photo activation, hybrid micelles, multidrug resistance, co mbined therapy

ABSTRACT

Currently, the simple integration of

multiple therapeutic agents within a single

nanostructure for combating multidrug resistance (MDR) tumors yet remains a challenge. Herein, we report a photo-responsive nanocluster (NC) system prepared by installing PDA nanoparticle clusters on the surface of TPGS (drug efflux inhibitor) micelles solubilized with IR780 (photosensitizer), in order to achieve combined chemo (CT)/photothermal (PTT)/photodynamic therapy (PDT) of drug-resistant breast cancer. Mediated by the fluorescence resonance energy transfer (FRET) and radical scavenging properties of PDA, NC shows prominently quenched fluorescence emission (~78%) and inhibited singlet oxygen generation (~67%) upon exposure to near-infrared


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(NIR) light (808 nm, 0.5 W cm-2), favoring a highly efficient PTT module. Meanwhile, the photothermal heat can also boost release of DOX whose intracellular accumulation can be greatly enhanced by TPGS. Interestingly, the first NIR irradiation and subsequent incubation (~24 h) can induce the gradual relocation and disintegration of PDA nanoparticles, thereby leading to activated PDT therapy under the second irradiation. Upon the temporally-controlled sequential application of PTT/PDT, the developed NC exhibited a great potential to treat MDR cancer both in vitro and in vivo. These findings suggest that complementary interactions among PTT/PDT/CT modalities can enhance the efficiency of the combined therapy of MDR tumor.

1. INTRODUCTION

Multidrug resistance (MDR), which induces the failure of chemotherapy (CT) treatment, is still recognized as one of the main obstacles for efficient cancer therapy. The overexpression of ATP binding cassette (ABC), like P-glycoprotein (P-gp) acts as


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an efflux pump to actively efflux drugs from tumor cells, resulting in insufficient therapeutically

efficacy.1-4

Corresponding

P-gp

inhibitors

like

D-α-tocopheryl

polyethylene glycol 1000 succinate (TPGS), have been devoted to inhibiting drug efflux.5 As a safe and amphiphilic pharmaceutical adjuvant, TPGS can form micelles in aqueous solution for MDR-overcoming therapy, where a simplified combination of the MDR inhibitor and therapeutic drugs can be achieved.6, 7 However, the disadvantages in premature drug release, low physicochemical stability, as well as complicated synthesis processes, limited the application of these micelle systems.8-10 Approaches toward combined therapy can be utilized to improve treatment efficiency, reverse the drug resistance, and reduce side effects. Near-infrared (NIR) light-activated phototherapies, including photothermal therapy (PTT) and photodynamic therapy (PDT), provide an alternative way for spatiotemporally controlled treatments of cancers via minimal invasion.11 When the toxic chemotherapy is combined with the targeting effect of phototherapy, the treatment is imparted with the ability of killing cancer cells only while avoiding the damage to normal tissues.12


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For PTT, photothermal conversion agents (PTCA) convert the electromagnetic energy of NIR light into heat to induce on-site hyperthermia and kill tumor cells.13, 14 However, a single photothermal therapy cannot achieve complete tumor eradication, owing to the heterogeneous distribution of heat over tumor tissues, and widespread concerns regarding the long term toxicity of widely employed inorganic PTCA.15,

16

Among the

recent exploitations, bioinspired polydopamine (PDA) is emerging as a biocompatible PTCA with mild aqueous synthesis requirements and relatively high photothermal conversion efficiencies.17-19 For PDT, appropriate photosensitizers (PS), as represented by lipophilic cation heptamethine dyes (e.g. IR780, IR820),20 generate singlet oxygen (1O2) or other reactive oxygen species (ROS) which can destroy cancer cells.21,

22

Micelles of amphiphilic polymers, especially those with MDR-overcoming functions, were typically used for the encapsulation of hydrophobic PS. However, PDT suffers from obstacles in cancer hypoxia and daylight phototoxicity during circulation.23

To overcome the problems mentioned above, many recent efforts have been made to realize the combination of PDT, PTT, and CT by codelivery of PTCA, PS, and


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anticancer drugs.24 Despite the recent strides in this multi-modal therapy, synergistic therapy on the basis of complementary interactions among three treatment modalities yet warrants further investigations in the state-of-the-art nanostructures with a facile integration of multiple therapeutic agents. For instance, very few systems addressed the challenges in simple co-loading of chemotherapeutic drugs and MDR inhibitors, shielding the PS from generating unwanted phototoxicity, as well as conveying the therapeutic modes responsively and/or temporally. In light of this, we envisioned that one of the reasonable solutions is to design disintegratable nanohybrids with multiple loading compartments and photoactivatable switches of therapeutic modalities. Noteworthily, PDA also possesses a great potential in the integration of nanostructures and guest molecules on multiple interfaces by its bioadhesive properties.25 Therefore, employing the merits of PDA in constructing nanohybrids is expected to be an ideal strategy to achieve a rational arrangement and an effective combination of functional entities.


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Herein, we report a photo-responsive nanocluster (NC) system on the basis of installing PDA nanoparticle clusters on the surfaces of TPGS-IR780 micelles to achieve trimodal synergistic therapy (CT, PDT, and PTT) of drug-resistant breast cancer, where PTT and photothermally triggered PDT were applied sequentially. As shown in Figure 1a, IR780 was loaded in the hydrophobic tocopheryl core of TPGS micelles (with sub100 nm sizes)26,

27

to function as the PDT sensitizer. Inspired by the strong interface

interactions (π-π stacking and hydrophobic interactions) of PDA toward aromatic molecules,28 PDA nanoparticles generated by self-polymerization were designed to be anchored/clustered in the hydrophilic poly(ethylene oxide) (PEO) shell of the micelles. A disulfide bond bearing crosslinker (cystamine) was introduced to crosslink PDA nanoparticles to impart the formed NC with a high stability. As demonstrated by systematic investigations, the clustered PDA nanoparticles thereafter play three essential roles for efficient phototherapy: (i) preventing the premature release of IR780; (ii) acting as a photophysical barrier to impede the fluorescence emission and ROS generation of IR780 for reducing phototoxicity during circulation; (iii) generating photothermal heat to trigger the dissociation of NC and activate PDT during the second


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NIR irradiation. Moreover, the PDA nanoparticles in NC can load doxorubicin hydrochloride (DOX) with a high payload (up to 1217 μg mg-1) and release it by multiple stimuli (weakly-acidic pH value, glutathione, and NIR) after efficient intracellular accumulation. The advantages of this simple and facile structural/function integration were then demonstrated in vitro and in vivo by the excellent synergistic efficacy in tumor ablation.

2. EXPERIMENTAL SECTION

2.1. Materials and Reagents

Ethanol (99.7%) was purchased from Fluka (Shanghai). TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate), dopamine hydrochloride (98%), cystamine dihydrochloride (Cys, AR) doxorubicin hydrochloride (DOX, 98%), reduced glutathione (GSH), tris(hydroxymethyl)aminomethane (TRIS, 99.9%) and 2-[4-(2-hydroxyethyl)-1piperazinyl]ethanesulfonic acid (HEPES) were obtained from Aladdin Industrial Inc


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(Shanghai). IR780 iodide was purchased from Sigma (Shanghai). Singlet oxygen sensor green (SOSG) was purchased from Thermo Fisher Scientific (Shanghai). DCFHDA (2,7-dichlorofluorescein diacetate) was purchased from Heowns Biochemical Technology Co., ltd (Tianjin). The DOX resistant human breast cancer (MCF-7/ADR) cell line was obtained from Bogoo Biological Technology Co., ltd (Shanghai).

Cell

staining dyes including calcein-AM/PI double stain kit, Hoechst 33258 were purchased from Yeasen Biological Technology Co., ltd (Shanghai). Cell counting kit-8 (CCK-8) was purchased from Dojindo Chemical (Shanghai).

2.2. Synthesis of TPGS-IR780@PDA Nanoclusters (NC)

To prepare NC, 280 mg of TPGS and 10 mg IR780 was firstly dissolved in 29 mL H2O and 2.9 mL ethanol, respectively. The above two solutions were then mixed by sonication, followed by the addition of 21 mg tris(hydroxymethyl)aminomethane (TRIS) and stirring for 30 min. Afterwards, dopamine hydrochloride (DOPA, 60 mg ) and cystamine dihydrochloride (Cys, 6 mg) was sequentially added and the reaction solution was stirred for 24 h at room temperature. Then the final product of the NC suspension


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was obtained by dialysis in water for three days. Finally, the morphology and structure of the NC was investigated by transmission electron microscope (TEM, JEOL 2010, Japan). The hydrodynamic size distributions and zeta potentials of the samples were obtained by a Zetasizer Nano instrument (Malvern, UK).

2.3. Photothermal Effect Evaluations

To evaluate the photothermal property, 1 mL of NC suspension (at different concentrations) loaded in a quartz cuvette was exposed to a near-infrared (NIR, 0.5 W cm-2, 808 nm) light. The temperature of solutions was recorded at an interval of 60 s by using a digital thermometer (HH806AU, Omega) with a thermocouple probe.

To evaluate the photostability, the NC solution was heated by applying the irradiaiton of the NIR light for 10 min, then cooled down to room temperature (in 15 min) by switching off the light source. The heating/cooling process was repeated three times. On the basis of the data from the cooling stage, the photothermal conversion efficiency (η) was calculated.


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To evaluate the photothermal effect invitro, the MCF-7/ADR cells were cultured in RPMI-1640 medium (GIBCO, New York) containing 10% fetal bovine serum (FBS, GIBCO), 100 U mL-1 of penicillin and streptomycin, and 1 µg mL-1 DOX in an incubation atmosphere (37 ℃ , 5% CO2). Then cells were seeded in a 24-well plate (3 × 105 cells/well) and cultured for 24 h. After that, the cells were treated with fresh medium, NIR irradiation (808 nm, 0.5 W cm-2, 10 min), NC (30 µg mL-1, 24 h of incubation) or NC (24 h) and subsequent NIR irradiation (NC + NIR). The temperature elevation in the wells was monitored using an infrared (IR) thermography camera (FLIR, E40, USA). Finally, the cells were washed with PBS for two times, and the double staining of calcein acetoxymethyl ester (calcein-AM, for alive cells)/propidium iodide (PI, for dead cells) was subsequently employed to evaluate cell death levels after different treatments. The stained cells were imaged by using an inverted fluorescence microscope (IX-71, Olympus).

2.4. Extracellular and Intracellular ROS Production by PDT


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The generation of extracellular ROS was evaluated with a singlet oxygen sensor green (SOSG) probe.29, 30 Here, SOSG at a concentration of 2.5 μM was incubated with the tested samples including TPGS-IR780 (2.1 μg mL-1) micelles, NC (30 μg mL-1), as well as NC after the first irradiation and continuous incubation of 24 h (NC + NIR + Incubation). After 10 min, the tested samples were irradiated by NIR for various time periods. Finally, the fluorescence intensities of oxidized SOSG by PDT-generated singlet oxygen were measured by a fluorescence spectrophotometer (RF-6000, Shimadzu) at an excitation wavelength of 504 nm.

The intracellular generation of ROS was detected by using a ROS-sensitive probe, i.e. 2,7-dichlorofluorescein diacetate (DCFH-DA), whose oxidation product is fluorescent. For the PDT involved groups, MCF-7/ADR cells were incubated with DCFH-DA (10 µM) at 37℃ for 30 min before the NIR irradiation. Finally, the fluorescence images of cells after different treatments, i.e. NC, NC + NIR, or NC + two sequential NIR irradiation (24 h of time interval) were acquired using a confocal laser scanning microscopy (CLSM, 510 META, Olympus). In the group of NC + two sequential NIR irradiation, the ROS


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probe was added before the second NIR irradiation. For the quantitative detection of intracellular ROS levels, the fluorescence of the cells were also analyzed by a flow cytometry (Accuri C6, BD).

2.5. Cell Uptake and Cytotoxicity Assay

To evaluate the cellular uptake of NC, cells were seeded onto confocal microscopy dishes (5 × 105 cells per dish) for 24 h. Subsequently, the culture medium was replaced with fresh medium containing 30 μg mL-1 of NC. After incubation for another 24 h, the cells were washed with PBS. After that, the cells were treated with 4% paraformaldehyde solution (0.5 mL) at 4℃ for 30 min. Finally, the cells were stained by Hoechst 33258 for 10 min and visualized by CLSM to observe the uptake of NC via the fluorescence from IR780.

The cytotoxicity was evaluated by a standard cell counting kit-8 (CCK-8) assay. The cells were cultured onto 96 wells (8 × 103 cells/well) and incubated for 24 h. Afterwards, the cells were treated with NC, NC-DOX or free DOX at different concentrations for another 24 h. Subsequently, NIR irradiation was applied in single application or


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sequential manner to evaluate the combination effect of phototherapy. Finally, 220 μL fresh medium containing 10% CCK-8 was added, and cells were further incubated for another 2 h. The cytotoxicity was measured by employing the absorbance at 450 nm via a microplate reader (Bio-Rad 680, USA).

2.6. Tumor Therapy In Vivo

The animal experiments were strictly conducted according to the regulations of the Institutional Animal Care and Use Committee of China with the approval of the ethics committee of Third Military Medical University (SYXK-PLA-20120031). Female nude mice (4 to 5 weeks, 20 ± 2 g) were purchased from the Animal Laboratory of Daping Hospital (Chongqing). For establishing a tumor model, MCF-7/ADR cells (2 × 107 cells with 20% Matrigel, BD Biosciences) were subcutaneously injected into the left flank near the mammary fat pat. When the tumor volume reached 60 mm3, the nude mice were randomly divided into 5 groups (5 mice per group): (a) saline, (b) DOX at a dose of 3 mg kg-1, (c) NC-DOX solution at an equivalent DOX dose of 3 mg kg-1, (d) NC at a dose of 3 mg kg-1 with two sequential NIR irradiation (24 h of time interval) and (e) NC-


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DOX solution at an equivalent DOX dose of 3 mg kg-1 with two sequential NIR irradiation (24 h of time interval). Mice were given intravenous injection treatments and the first/second NIR was applied 6 h/30 h after injection. The day when treatments started was set as day 0. The body weights and the tumor volumes were measured every 2 days. These treatments period lasted for 21 days. The formula (Volume (mm3) = length (mm) / 2 × width2 (mm)) was used to calculate the tumor volume. Subsequently, all the mice were sacrificed and the major organs (heart, liver, spleen, lung, and kidney) and tumor tissues were harvested for hematoxylin and eosin (H&E) staining assay.

To study the levels of proliferation and apoptosis, immunofluorescence staining was used. The tumor samples were collected and fixed for transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) staining assay. Besides, caspase-3 immunofluorescence staining was performed to measure the apoptosis degree in the heart issue slices from each group. The slices were examined with CLSM.

2.7. In Vivo Photothermal Therapy


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The increase of temperature at the tumor region upon NIR (0.5 W cm-2, 5 min) irradiation was evaluated by an infrared (IR) thermography camera (E40, FLIR, USA). Typically, 100 μL of NC (3 mg kg-1) or 100 μL of saline was intravenously injected to the tumor of mice. The temperature at the tumor region was measured at different postinjection time-points (1, 3, 6, 9, 18, 30 h).

2.8. Nanoparticle Biodistribution and Drug Accumulation

To investigate the distribution of NC in MCF-7/ADR tumor-bearing mice, the fluorescence signal of IR780 in vivo was detected by a IVIS Lumina Ⅲ imaging system (Perkin Elmer, USA). NC (3 mg kg-1) was intravenously injected into MCF-7/ADR tumorbearing mice. The whole body fluorescence imaging was taken at different postinjection time-points (6, 18, 30 h). Then, IVIS Living Imaging Software was applied to analyze the images.

To evaluate drug accumulation degree of NC within tumors, mice bearing MCF7/ADR tumor were randomly divided into 2 groups: (a) DOX at a dose of 3 mg kg-1, (b) NC-DOX solution at an equivalent DOX dose of 3 mg kg-1. Mice after different


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treatments were sacrificed at different time-points post injection (6, 18, 30, 36 h). Tumors were collected, washed, and weighed.

Next, the tumor tissues were

homogenized, and DOX was extracted with 200 μL of isopropyl alcohol containing 0.5% acetic acid. Finally, the concentration of DOX was measured by using high-performance liquid chromatography (HPLC, Agilent 1260).

3. RESULTS AND DISCUSSION

For the preparation of NC, amphiphilic TPGS molecules were firstly self-assembled above the critical micelle concentration (CMC) of 2 × 10−4 M to form micelles and solubilize hydrophobic IR780.31 Transmission electron microscopy (TEM) analysis showed the TPGS-IR780 micelles are ~70 ± 20 nm (Figure 1b) in size. To prepare PDA nano-clustered TPGS-IR780 micelles, dopamine (DOPA) was self-polymerized in weakly basic aqueous solution, followed by nucleation and growth of PDA nanoparticles on the surface of the micelles to form hybrid nanoclusters. Concomitantly, the PDA nanoparticles inside the nanoclusters were crosslinked by cystamine dihydrochloride


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(Cys) by the Michael addition/Schiff base reactions of PDA.32-34 The obtained NC (TPGS-IR780@PDA) particles possess an average size of 90 nm (Figure 1c) and a clustered structure of PDA nanoparticles (5-10 nm). The average hydrodynamic size of NC is around 122 nm with a low polydispersity index (PDI) of 0.02, which indicated the uniformity of the particles (Figure 1c, inset). Notably, the synthesis in the absence of both TPGS and IR780 led to irregular shaped PDA particles without the cluster structure, confirming the template effect of micelles in generating PDA clusters. Moreover, the absence of IR780 resulted in microscale aggregates (Figure S1), possibly as a consequence of the lack of sufficient PDA-micelle interactions via the π-π stacking interactions between aromatic rings of IR780 solubilized in the hydropic core and PDA on the hydrophilic PEO shell of the micelles.28 Stronger crosslinking of PDA by employing higher molar ratios of Cys/DOPA also led to the diminished clustering. We also found that 17%/33% increase in the amounts of DOPA and Cys resulted in smaller NC particles (~35 nm, Figure S2a and inset) with large interparticle space of PDA, as well as a complete disappearance of the clustered structure (Figure S2b). This might be


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related with the extraction of IR780 from micelles to PDA’s surfaces by the largely accumulated PDA particles via π-π stacking interactions.

The suspension of PDA nanoparticles has a broad-spectrum absorption from UV to NIR regions, while IR780 (dissolved in EtOH) exhibits a strong absorption peak at 780 nm (Figure S3a), beneficial for their PTT and PDT applications under NIR irradiation. TPGS-IR780 micelles and NC dispersion present a similar absorption peak at 790 nm, indicative of the IR780 loading. The 10 nm red-shift of the peak can be explained by the hydrophobic interaction inside micelles.35 The loading content and encapsulation efficiency of IR780 were calculated to be 4.5% and 89.8%, respectively. The coexistence of TPGS and PDA in NC was confirmed by the FTIR spectra (Figure S3b).

The formation of PDA nanoclusters on TPGS-IR780 micelles enabled us to control the physicochemical properties of NC by NIR and GSH triggers: (i) the soaring heat locally generated by photothermal conversion may lead to the conformational changes of TPGS’s polyethylene oxide (PEO) segments in terms of the decrease in both the hydrophilicity and the overall size, and in turn the relocation of the PDA nanoparticles


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interspersed around the PEO chains; (ii) the disulphide bond between PDA nanoparticles

maybe

cleaved

responsively

by

intracellular

redox

conditions.

Interestingly, the clustered PDA nanoparticles were dislocated and rearranged (Figure S4a) after NIR irradiation (808 nm, 0.5 W cm-2, 10 min), followed by a slow progress of cluster disintegration after incubation for 12 h and 24 h (Figure S4b and Figure 1d). The progressive change was also verified by the continuous reduction of the zeta potentials from 0 mV to -17 mV (Table S1). However, the structural stability of NC was maintained without the NIR trigger. The particle size of the NC did not significantly change after 48 h in cell culture media or simulated body fluid (SBF), and the suspension of NC remained clear without any precipitation, indicating their great structural integrity (Figure 1e, inset). Additionally, the morphological evolution of NC by the presence of 10 mM GSH was much slower, and particles remained almost intact after 24 h (Figure S4c). After a significantly longer incubation time of 72 h, the structure of NC was partially disintegrated to necklace-like nanostructures with different lengths (Figure S4d). These results suggest that the disintegration of NC is more sensitive to the photothermal stimulus.


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In

addition

to

the

physicochemical

characteristics,

the

photophysical

and

photochemical properties of NC may also be tailored with NIR stimulus. The intrinsic fluorescence signals of IR780 were reduced (by ~28%, Figure 1f) due to the quenching effects of PDA in terms of light absorption (Figure S3a) and the fluorescence resonance energy transfer (FRET).36 After applying NIR, the fluorescence of IR780 was significantly quenched to ~22% and 11% for NC and TPGS-IR780 micelles, respectively, due to a tighter aggregation of IR780 in the micelle core induced by photothermal heating. Notably, the fluorescence of TPGS-IR780 micelles can not be recovered during further incubation. This can be explained by light-induced decomposition (i.e. photobleaching) of IR780 resulting in the loss of fluorescence and photoactivity, with which the ROS generation may be related.37 In comparison, the fluorescence of NC can be recovered in a process of a steep ascent at the first stage and then a slow increase to a stable value (close to that of NC without NIR irradiation), implying that NC can protect IR780 from photo-decomposition. As previously reported, PDA possesses excellent free-radical-scavenging property,38,

39

which might be responsible for the

photo-protection effect of the PDA nanoparticles adjacent to the IR780 dyes.


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Additionally, the fluorescence maintained with/without the presence of GSH if there was no NIR stimulus introduced.

Motivated by the interesting photo-activatable structural evolution, we then investigated the photothermal properties of NC before and after the disintegration. As shown in Figure 2a and Figure S5a, NC displayed a temperature elevation of ~20°C at a concentration of 30 µg mL-1 upon exposure to the NIR light (808 nm, 0.5 W cm-2, 20 min). Moreover, the temperature increase followed a concentration- and timedependent manner during irradiation, indicating that NC can rapidly and efficiently convert the electromagnetic energy of NIR light into heat. By contrast, an equivalent IR780 concentration of 2.1 μg mL-1 led to a temperature increase of only 10°C for TPGS-IR780 micelles. Moreover, the temperature increased by 24°C in 7 min and then decreased gradually at a higher IR780 concentration of 30 μg mL-1 (Figure S5b), because of the photo-decomposition in the PDA-free micelles. The temperature elevation of TPGS-IR780 micelles decreased by 7°C in the second cycle. In comparison, NC suspension at both high (200 µg mL-1) and low (30 µg mL-1) concentrations


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exhibited slight changes (~2°C) in temperature elevation after three repeated cycles of NIR laser irradiation (Figure 2b), revealing a stable photothermal effect in a short term. However, when the NC suspension after applying the first NIR irradiation was continuously incubated at 37°C for 24 h, the photothermal effect was significantly weakened (6.3°C of temperature elevation, Figure 2c), possibly as a result of the structural disintegration. The calculated photothermal conversion efficiency (η)40 of NC was 22.2% (Figure S5c, d), which is 1.7 fold larger than that of TPGS-IR780 micelles (13.3%, Figure S5e, f). This indicates that the combination of PDA and IR780 in the structural form of NC contributed to the enhanced photothermal conversion. Therefore, the responsive structure change can weaken the combination and alter the photothermal modality.

The influence of structural disintegration on the photodynamic modality of NC was then investigated. SOSG, a specific probe whose reaction with ROS can generate fluorescent SOSG endoperoxides (SOSG-EP) emitting at 525 nm,29 was employed to evaluate the photodynamic activity. As shown in Figure S6a-c, the fluorescence levelled


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off after ~20 min of irradiation. The ability of NC to generate ROS upon light exposure was remarkably (~67%) lower than TPGS-IR780 micelles (Figure S6a, b), which can be explained by the protection effect of PDA in term of the ROS scavenging.38, 39 However, the photodynamic activity of NC can be recovered and reach 83% of the PDA-free micelles when NC after the first NIR irradiation was continuously incubated for 24 h (Figure S6c and Figure 2d). Furthermore, the recovering process was time-dependent (Figure 2e). The strongest fluorescence intensity was obtained when the time interval between two NIR irradiations was ~24 h. In comparison, the ROS generation capacity of NC at different incubation times remained unchanged in the absence of pretreatment with the first NIR irradiation. This can further support that the gradual recovery of the photodynamic activity was mediated by the photo-activated structural disintegration. The results above suggest the design NC system possesses a novel photo-responsive property of PTT/PDT switching by sequentially applying two NIR irradiations. To impart NC with chemotherapy modality, DOX was successfully loaded in NC by adsorption on PDA via the π-π stacking and hydrophobic interactions.18 The loading capacity reached 1217 μg mL-1 as a consequence of the clustered structure, and the release can be


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triggered by multiple stimuli of pH value, glutathione (GSH) and NIR (Figure S7 and Figure 2f). As shown, the obtained release profiles show a cumulated DOX release degree of about 13%, 25%, and 54% after 72 h of incubation at pH 7.4, pH 5.0, pH 5.0 with GSH, respectively, while the release degree was increased by ∼43% upon the simultaneous application of all stimuli (pH, GSH, NIR) (Figure 2f). The release behavior is in agreement with previous reports on DOX-loaded PDA nanoparticles.18

To test the delivery properties of the nanoassemblies in vitro, we examined the cellular uptake of NC and the delivery of DOX under confocal laser scanning microscopy (CLSM). As shown in Figure 3 a1 and Figure S8 a1, the fluorescence signal of IR780 was dispersed in the cytoplasm after 24 h of incubation, implying a good uptake of NC in MCF-7/ADR cells. Compared with the control MDR cells incubated with DOX (10 μg mL-1) only, cells incubated with NC (30 μg mL-1) showed effectively suppressed P-gp expression (~35%, Figure S9) which can be further reduced to 50% by NIR irradiation. These results suggest a remarkable role of the delivered TPGS in inhibiting drug efflux. We next evaluated the intracellular drug accumulation of free DOX,


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NC-DOX or NC-DOX + NIR (Figure 3 a2-a4 and Figure S8 a2-a4). Obviously, the group of NC-DOX + NIR showed stronger DOX ﬂuorescence signal than other groups, indicating that the photothermal heat can also boost the release of DOX.

To evaluate the consequent PTT efficacy after the effcient uptake and drug efflux inhibition, fluorescence images of cells costained with calcein acetoxymethyl ester (calcein-AM) and propidium iodide (PI) were employed to confirm the killing efficiency of cancer cells after different treatments. The cells were treated with NC (30 μg mL-1) or NIR irradiation showed strong green signals, indicating the absence of apparent cell death (Figure 3 b1–b3 and Figure S8 b1–b3). In contrast, the cells showed strong red fluorescence after NC incubation and NIR irradiation (Figure 3 b4 and Figure S8 b4), implying that severe cell death was induced by NC via photothermal effect. Meanwhile, as indicated by the thermal images of cells, the temperature of the NC + NIR group was significantly higher than that for other groups. The temperature was elevated by 12.5°C after 10 min of NIR irradation, which can effectively kill cancer cells.41, 42


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In light of NC’s photo-responsive property of photothermal/photodynamic switching by sequentially applying two NIR irradiations, the ability of NC to generate intracellular ROS for PDT was then explored in vitro. MCF-7/ADR cells were stained with a intracellular ROS probe, i.e. DCFH-DA before CLSM imaging. No obvious green fluorescence was found in NC group after 24 h of incubation, as well as the control group without any treatment (Figure 3 c1, c2 and Figure S8 c1, c2). Weak green signals appeared in NC treated cells after the first laser irradiation (Figure 3 c3 and Figure S8 c3) owing to the photo-protection effects of PDA on IR780 dyes, which is in agreement with the previous ROS-detection results in the absence of cells. Notably, the NC + two sequential NIR (24 h of time interval) group showed a significantly strong fluorescence (Figure 3 c4 and Figure S8 c4). The flow cytometry (FCM) analysis (Figure 3 c1-c4, inset; Figure S8 c1-c4) displayed the similar trend as that of CLSM results, further proving that the disintegrated NC after the first irradiation and the subsequent incubation produced a large amount of single oxygen during the second irradiation.


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The efficicacies of combination therapies with NC were then investigated. The cytotoxicity was evaluated by the cell counting kit-8 (CCK-8) assay (Figure 3 d1-d4). The viability of cells remained above ~82%, even they were cultured with NC at a high concentration of 100 μg mL-1 , implying a good biocompatibility. By contrast, the viability of cells treated with NIR for 5 min or 10 min dramatically decreased as a function of NC concentration, and reached ~26% or ~11%, respectively, at the highest concentration of 100 μg mL-1 (Figure 3 d1). At a lower NC concentration of 30 μg mL-1, continuous incubation resulted in significantly reduced viability from the first (~52%) to the second NIR irradiation (~21%) (Figure 3 d2), indicating the combined therapeutic efficacy of PDT and PTT. In addition, the cytotoxicity of NC-DOX + NIR group also showed a dosedependent increment. The cell viability of the NC-DOX + NIR group at the highest DOX concentration of 100 μg mL-1 decreased to ~2%, which was remarkably lower than that of the cells treated with an equivalent amount of free DOX (~70%) and NC-DOX (~30%) (Figure 3 d3). These results illustrate that NC possesses the ability to combine CT and phototherapy treatments. As shown in Figure 3 d4, at a lower DOX concentration of 37 μg mL-1 (with an equivalent NC concentration of 30 μg mL-1), continuous incubation


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resulted in significantly reduced viability from the first (~29%) to the second NIR irradiation (~10%). Thus, it is clear that the temporally-controlled combination treatment can generate synergistic cytotoxicity.

Encouraged by the exciting results in our combined treatment of triple modal in vitro, we then evaluated the in vivo antitumor activity of NC on mice bearing MCF-7/ADR tumors (Figure 4). Firstly, photothermal therapy was applied. At different times postinjection of NC, an IR thermography camera was used to record the increase of temperature at the tumor region, where a maximum was observed ~6 h postinjection (Figure 4a). At this time point, the temperature of the tumor region injected with NC exhibited a rapid increase from 35.5°C to 46.5°C (Figure 4b and c) after 5 min of irradiation, whereas only a very slight elevation was detected in the saline group. The results indicated that NC could generate enough hyperthermia upon the NIR laser irradiation at 6 h post-injection.

By utilizing the fluorescence imaging property of IR780, the NIR fluorescence imaging in the mice were captured to evaluate distributions of NC in mice. As demonstrated in


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Figure 4d and Figure S10, obvious fluorescence signals in tumor were clearly seen at the same time point of 6 h. Moreover, the distribution of NC in tumor increased significantly with time, and the strongest fluorescence was observed at 30 h postinjection, owing to the well-known EPR (enhanced permeability and retention) effect of nanoparticles in tumors and the possible redox-responsive disassembly of NC under in

vivo conditions. Relatively strong IR780 fluorescence was also observed in the liver, lung and kidney because of the biodistribution. Quantitative analysis of DOX in the tumor tissue at different time points post injection (6, 18, 30 and 36 h, Figure S11) indicated higher accumulation of DOX in tumors for NC-DOX. At 36 h post injection, the DOX accumulation (1.1 % ID/g) was 3.7 times larger than that of free DOX. These data indicated that NC-DOX could effectively enhance the accumulation of DOX in the tumor tissue, possibly owing to the nanoparticle-mediated delivery43 and TPGS-inhibited drug efflux.

Next, the strategy of combined therapy was employed and investigated, where the treatments were divided to 5 groups: saline, free DOX, NC-DOX, NC + two sequential


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NIR (NC + NIR), and NC-DOX + two sequential NIR (NC-DOX + NIR). Ten injections were conducted within the 21 days, at time intervals of 2 days. To apply the time sequence parameters obtained from the in vitro studies, the NC or NC-DOX group was exposed to NIR laser irradiations 6 h post injection and 24 h after the first irradiation. As presented in Figure 4e, the body weights of mice showed no significant weight loss, indicating the negligible systematic toxicity. Intravenous injection of free DOX could only slightly inhibit the growth of the drug-resistant tumor (Figure 4f). In comparison, the inhibition was much more effective for the NC + NIR and NC-DOX + NIR groups (**p

Photodynamic and Combined

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