br Subsequently the cytotoxicity of BP DACHPt in
Subsequently, the cytotoxicity of BP/DACHPt in air-exposed water after 72 h was quantified with the MTT assay. As displayed in Fig. 3B and S13, BP/DACHPt is cytotoxic against HeLa ATPγS tetralithium salt in a concentration-dependent manner without NIR laser irradiation. After irradiation, the survival rates of HeLa cells treated with 20 μg/mL and 50 μg/mL BP/ DACHPt were merely 32% and 5% respectively. Notably, this complex was also photothermally cytotoxic towards other tumor cells (Fig. S14). In addition, the photothermal toxicity of freshly prepared BP/DACHPt was stronger than that of bare BP, which must be attributed to the chemotherapeutically cytotoxicity (Fig. S15). Thus, the chemother-apeutic toxicity of BP/DACHPt against HeLa cells was thereafter tested in detail. Although this complex was not as toxic as antitumor agent DACHPtCl2, the toxicity was significantly enhanced with prolonged culture time (Figs. 3C and S16), probably owing to sustained release of DACHPt. After 48 h of culture, the IC50 value reached 41.6 μg/mL (Table S1). In consideration of that the mass of DACHPt in BP/DACHPt was twice that of BP, the loaded DACHPt would induce considerable chemotherapeutic toxicity while 20 μg/mL BP was markedly photo-thermally cytotoxic.
3.5. Apoptosis mechanism study
Then the mechanism by which BP/DACHPt killed tumor cells was unraveled by flow cytometry. HeLa cells were treated with BP/DACHPt for 24 h in the presence or absence of NIR laser irradiation, and then labeled with Annexin V and PI. The Annexin V+-PI− cells mean early apoptosis occurred, and the Annexin V+-PI+ ones underwent late apoptosis/necrosis . Fig. 4 presents that BP/DACHPt kills 37% of the cells without irradiation, basically through early apoptosis as a result of chemotherapy. After being irradiated, the cells were almost completely killed, verifying that irradiation significantly promoted late apoptosis/necrosis (60.2%), which can be ascribed to photothermal toxicity like that reported before . Collectively, BP/DACHPt was capable of simultaneous photothermal therapy and chemotherapy.
3.6. In vivo antitumor eﬃcacy
At last, we evaluated the in vivo antitumor eﬀects and tissue safety of BP/DACHPt. Female sever combined immunodeficient (SCID) mice with HeLa cell xenografts were treated by saline + NIR, bare BP + NIR, BP/DACHPt, or BP/DACHPt + NIR through intratumor injection. Both bare BP and BP/DACHPt were predispersed in air-exposed water for 72 h. NIR represents the tumor sites were irradiated with 808 nm NIR laser at 1.5 W/cm2 for 10 min. At day 14, the tumor growth of the BP/ DACHPt + NIR group was totally inhibited after NIR laser irradiation, even leading to tumor ablation (Fig. S17). Contrarily, the tumor growths of saline + NIR and bare BP + NIR groups were unaﬀected, and that of the BP/DACHPt group was only partly suppressed, because of the chemotherapeutic eﬀects of DACHPt. As expected, BP/DACHPt was highly photothermally stable, being capable of eﬃcient Chemical Engineering Journal 375 (2019) 121917
photothermal therapy and chemotherapy simultaneously.
We thereafter tested the in vivo performance of PEG-coated BP/ DACHPt (BP/DACHPt-PEG) through intravenous injection. With good stability in vitro, BP/DACHPt-PEG showed prolonged blood circulation as expected (Fig. S18A) and eﬃcient tumor accumulation after 12 h of injection (Fig. S18B), similar to other reported nanosheet-based de-livery systems . The mice were treated by Saline (1), NIR (2), BP/ DACHPt-PEG (3), or BP/DACHPt-PEG + NIR (4) through intravenous injection. After 12 h of injection, the temperature changes of tumor sites were monitored using IR thermal camera. As a consequence, the tumor temperature of the 4 group was significantly raised to 52.7 °C, but those of the other three groups barely varied (Fig. 5A and B). The complete ablation of tumor after NIR laser irradiation also confirmed the ex-cellent anticancer eﬃcacy of BP/DACHPt-PEG through intravenous injection (Fig. 5C and D). As evidenced by the histopathological assay, the 4 group underwent the most severe tumor necrosis (Fig. 6A). Fur-thermore, both body weight and histopathological assay proved negli-gible side eﬀects on the mice (Figs. 5E and 6B). Taken together, BP/ DACHPt-PEG had excellent photothermal and chemotherapeutic eﬀects as well as biocompatibility in vivo.
In summary, we developed a novel drug-self-stabilized BP-based drug delivery system. BP nanosheets loaded the active species DACHPt twice their weight to form the coordination complex BP/DACHPt, thereby significantly boosting the stability during long-term dispersion in air-exposed water and maintaining the photothermal performance. BP/DACHPt with ultra-high drug loading capacity underwent acid- and NIR-responsive release of intact DACHPt, thus inducing evident NIR photothermal and chemotherapeutic cytotoxicities in vitro. Finally, this complex exerted tumor ablation in vivo based on the combined photo-thermal and chemotherapeutic eﬀects. Thus this simple strategy of using drug itself to construct stable BP-based drug delivery system, opens a promising avenue for clinical application of BP in cancer therapy.