Sequential PDT and PTT Using Dual‐Modal Single‐Walled Carbon Nanohorns Synergistically Promote Systemic Immune Responses against Tumor Metastasis and Relapse

Abstract Immune responses stimulated by photodynamic therapy (PDT) and photothermal therapy (PTT) are a promising strategy for the treatment of advanced cancer. However, the antitumor efficacy by PDT or PTT alone is less potent and unsustainable against cancer metastasis and relapse. In this study, Gd3+ and chlorin e6 loaded single‐walled carbon nanohorns (Gd‐Ce6@SWNHs) are developed, and it is demonstrated that they are a strong immune adjuvant, and have high tumor targeting and penetration efficiency. Then, three in vivo mouse cancer models are established, and it is found that sequential PDT and PTT using Gd‐Ce6@SWNHs synergistically promotes systemic antitumor immune responses, where PTT stimulates dendritic cells (DCs) to secrete IL‐6 and TNF‐α, while PDT triggers upregulation of IFN‐γ and CD80. Moreover, migration of Gd‐Ce6@SWNHs from the targeted tumors to tumor‐draining lymph nodes sustainably activates the DCs to generate a durable immune response, which eventually eliminates the distant metastases without using additional therapeutics. Gd‐Ce6@SWNHs intervened phototherapies also generate durable and long‐term memory immune responses to tolerate and prevent cancer rechallenge. Therefore, this study demonstrates that sequential PDT and PTT using Gd‐Ce6@SWNHs under moderate conditions elicits cooperative and long‐lasting antitumor immune responses, which are promising for the treatment of patients with advanced metastatic cancers.

Synthesis and Characterization of Gd-Ce6@SWNHs. Gd-Ce6@SWNHs was synthesized according to our previous report. [2] Briefly, to modify pristine SWNHs, PEG-PLGA (10 mg) and C 18 PMH (10 mg) were mixed into 1 mL of THF and then added dropwise into 1 mL of aqueous SWNHs suspension (1 mg mL -1 ). The mixture was sonicated at a cold water-bath with nitrogen bubbling for half an hour to evaporate THF. And then kept sonicating for another 1-2 hours to yield a black suspension. The suspension was centrifuged at 10,000 rpm for 30 min to remove any large SWNHs aggregates. Then the dual-polymer-coated SWNHs were collected by ultrafiltration (Amicon Ultra-4, MWCO 100 K, Merck Millipore, Darmstadt, Germany).
The mixture was stirred for 1 h at room temperature and then sonicated for an additional 1 h. The excess Ce6 was removed by dialysis (Mw~8,000-14,000) against deionized water overnight. The drug-loading capacity (DLC) and drug-loading efficiency (DLE) were evaluated by the fluorescence signal of Ce6@SWNHs dialysate against known standards spectrum of free Ce6 (Ex=408 nm/Em=650 nm).
The hydrodynamic sizes and zeta potentials were examined by dynamic light scattering (DLS, Malvern Zetasizer Nano ZSP, Malvern, United Kingdom) in deionized water at 25°C. Transmission electron microscopy (TEM) images were obtained from JEM-2011 (JEOL, Japan) at 200 kV. NIR absorption spectra were recorded with a NanoDrop 1000 (Thermo Scientific, Wilmington, USA). Excitation and emission spectra of nanosystems were evaluated by a fluorescence spectrophotometer (F-2700, HITACHI, Tokyo, Japan), and the BET specific surface area was detected by automated gas sorption analyzer (Autosorb, iQ2, Quantachrome Instruments, FL, USA). The concentrations of gadolinium were measured by ICP-MS (Z-2000 series, HITACHI, Tokyo, Japan) and then T1 relaxivity of Gd-Ce6@SWNHs was determined by linear fitting the reverse T1 relaxation times measured by an NMR analyzer (Mini spec mq60, Bruker, Karlsruhe, Germany) as a function of Gd 3+ concentrations (in mM).
Phototherapy using Gd-Ce6@SWNHs in vitro. To assess the photothermal performance of Gd-Ce6@SWNHs, the lyophilized nanoparticles were resuspended into 400 µL of deionized water at different concentrations (0, 5, 10, 20, and 50 µg mL -1 in SWNHs) and then illuminated with an 808 nm laser at a power intensity of 1 Wcm -2 or irradiated at different power densities (0.5, 1, 1.5 and 2 W cm -2 ). A digital thermometer was used to monitor temperature change with a thermocouple probe submerged in the solution. To test the thermal stability, Gd-Ce6@SWNHs (20 µg mL -1 ) was alternatingly irradiated for six times at the power density of 1 W cm -2 for 5 min. The digital infrared-thermal photos were captured by IR thermal camera (FOTRIC, Shanghai, China). For analysis of the photothermal conversion efficiency (η) under 808 nm laser irradiation, correlation equations were applied in the above-recorded data according to the previous report. [5][6] Where ℎ is the transfer coefficient, is the surface area of the container, at a density of 40 mW cm -2 . For PTT, the cells were irradiated by an 808 nm laser for 5 min at different power densities (1, 1.5 and 2 W cm -2 ). For PDT + PTT combination therapy, the cells were exposed to the 650 nm laser at the power density of 40 mW cm -2 and the 808 lasers at 1.5 W cm -2 for 5 min sequentially. After the treatments, cells were supplemented with fresh media and further maintained for an additional 24 h. Finally, the cell viability was evaluated by CCK-8 assay as described previously. [7][8] Accordingly, the half-maximal inhibitory concentration values (IC 50 ) of PDT, PTT, or PDT + PTT combination therapy were determined from dose-response curves. The combination index (CI), which indicates the type of anticancer mechanism of the combined therapy, was calculated according to the formula = Where 1 and 2 are the concentrations of the first and the second therapeutic required to achieve a certain effect in combination therapy, and 1 and 2 are the concentrations of the first and the second drugs that generate an identical effect alone. When < 1, = 1, or > 1, the two therapeutics are implied to have synergistic, additive, or antagonistic effects, respectively. [9][10][11] The therapeutic effects of PTT and PDT were also evaluated by immunohistological chemistry. For PTT, 4T1 cells were co-incubated with Gd-Ce6@SWNHs (10 μg mL -1 ) for 12 h and irradiated with an 808 nm laser (1.5 W cm -2 ) for 3, 5, and 7 min. Live-dead cell staining was performed by Calcein-AM/PI staining kit (Cat: 40747ES76, Yeasen Biotech Co., Ltd., Shanghai, China). Namely, the live cells could be penetrated by the green fluorescent dye (Ex/Em = 488/518 nm).
However, since the dead cells could be simply stained with pyridinium iodide (PI), a red fluorescent dye which could hardly penetrate the live cell membrane (Ex/Em = 488/615 nm). After staining, the cells were examined using a dark-field fluorescence microscope ECLIPSE Ti-E (Nikon, Tokyo, Japan). Singlet oxygen generation after PDT was determined by the DCFH-DA assay. [12][13] The DCFH-DA is sensitive to SO and can be oxidized to a strong green fluorescent substance, dichlorofluorescein To further demonstrate the immunogenicity induced by phototherapy, activation of DCs was evaluated using a transwell system. The immature DCs (iDCs) were first derived from the bone marrow of 8-week-old Blab/c mice according to the previous reports. [14] The iDCs were incubated with Gd-Ce6@SWNHs or PEGylated SWNHs at For hematoxylin and eosin (H&E) examination, the lung, major organs, and tumors were harvested at the predetermined time and fixed in 4% neutral buffered formalin, embedded in paraffin and processed for H&E (Aladdin, Ontario, CA) staining according to the protocol provided by the manufacturer and previous reports. [8,17] Detection of lung metastases. The lungs of mice were injected with India ink through the trachea and fixed with Fekete′s solution according to the previous report. [2,18] For histological examination of the metastases, the lungs were dissected, embedded in paraffin and processed for H&E staining. Tumor metastasis sites subsequently appeared as white nodules in the digital photograph or as the bulk of aggregates in H&E slices. The nodules were counted under a digital microscope.