br Next we also visually
Next we also visually observed the stability of BP/DACHPt that was fabricated by coordinating micro-sized BP sheets with DACHPt. By using AFM, Castellanos-Gomez et al. found that dense droplets appear on the surface of BP after exposure to humid air because of BP oxidation . Being kept in air with 95% humidity at room temperature for 24 h, bare BP indeed formed fairly dense droplets on the surface (Figs. 2G and S6). On the contrary, the surface of BP/DACHPt sheet hardly changed (Figs. 2H andS6). The optical results also demonstrated the robust stability of BP/DACHPt (Fig. S7). These results directly proved that the coordination between DACHPt and BP eﬀectively in-hibited the BP oxidation in humid air, thereby boosting its stability.
Then PEG-coated BP/DACHPt (BP/DACHPt-PEG) was also prepared by PEGylation of BP nanosheets with PEG-NH2 like previous report . The amount of coated PEG on the BP surface was quantified as
Fig. 4. Flow cytometry analysis of cells incubated with BP/DACHPt in the absence or presence of NIR irradiation.
about 34% (w/w%) of BP nanosheets by ICP-MS. Without obvious size changes for 48 h (Fig. S9), BP/DACHPt-PEG was proved to be stable in phosphate-buﬀered saline (PBS) and DMEM cell culture medium con-taining 10% FBS, which is quite diﬀerent from BP and BP/DACHPt (Fig. S8). Along with the inhibited degradation under ambient condition, BP/DACHPt-PEG would be a stable drug delivery system for cancer therapy.
3.3. Drug release behavior study
Based on the above experimental results, we then evaluated the drug release from BP/DACHPt under diﬀerent conditions (pH 7.4 and 5.0) by measuring Pt levels in solution with ICP-MS. As the active species of a potent antitumor drug, DACHPt under release as required would probably synergize with photothermal therapy agent BP to combat tumors more eﬀectively. After 48 h of culture, 16.1% of DACHPt was released at pH 7.4, whereas 35.4% was released at pH 5.0 (Fig. 2I). At pH 7.4, DACHPt was released from BP/DACHPt by sub-stitution with chloride ions instead of BP in PBS . Since the zeta potential of BP and BP/DACHPt became more neutral or even slightly
positive in acidic conditions (Fig. S10), the accelerated release may be ascribed to the increased FSL1 of hydrogen ions on BP, further weakening the coordination between DACHPt and BP . Further-more, brief NIR laser irradiation (10 min) was able to induce momen-tary burst release. The NIR-induced burst release should result from the raising of the local temperature by BP through photothermal eﬀects, which accelerated the movements of drugs, being in accordance with the previous literature . Besides, the drug release under various conditions was validated by HPLC-MS to ensure that the compound could still exert antitumor eﬀects. The molecular weights of DACHPt released from BP/DACHPt in PBS and BP/DACHPt at pH 5.0 after NIR irradiation were both the same as that of DACHPt in the original so-lution (Fig. S11). Accordingly, the acid- and NIR-responsive BP/ DACHPt was capable of targetedly delivering DACHPt to tumors while being intact. Based on facile preparation, stable performance and ultra-high drug loading capacity, BP/DACHPt is undoubtedly a feasible drug delivery system.
Fig. 5. In vivo antitumor eﬀect of BP/DACHPt-PEG. A) IR images of HeLa tumor-bearing mice (1: Saline; 2: NIR; 3: BP/DACHPt-PEG; 4: BP/DACHPt-PEG + NIR). B) Time-dependent temperature increase at tumor sites. C) Relative tumor growth after diﬀerent treatments. D) Representative images of harvested tumors from each group. E) Body weight changes of each group at day 14.
Fig. 6. H&E stained (A) tumor slices from mice at day 14 after treatment with Saline, NIR, BP/DACHPt-PEG, BP/DACHPt-PEG + NIR and B) heart, lung, liver, spleen, and kidney slices from mice at day 14 after treatment with BP/DACHPt-PEG and BP/DACHPt-PEG + NIR via intravenous injection (scale bar = 100 μm).
3.4. In vitro photothermal experiments and cytotoxicity assays
To assess the in vitro cytotoxicity of BP/DACHPt, we compared the killing eﬀects of bare BP and BP/DACHPt pretreated in air-exposed water for diﬀerent time points on HeLa cells by fluorescence micro-scope. BP nanomaterials have well-documented remarkable photo-thermal antitumor eﬀects . After 10 min of irradiation with 808 nm NIR laser (power density: 1.0 W/cm2), the cells were co-stained by calcein AM (green fluorescence, live cells) and propidium iodide (PI, red fluorescence, dead cells). As a result, freshly prepared bare BP and BP/DACHPt at 50 μg/mL managed to kill almost all tumor cells (Fig. S12). However, bare BP no longer worked well after 72 h in air-exposed water (Fig. 3A), indicating that it had basically been all degraded. In contrast, BP/DACHPt could still kill tumor cells, depending on the long-term stable photothermal performance in the external environment, being in agreement with the in vitro stability results mentioned above.