br Fig Fabrication and characterization of BP DACHPt A Surfa
Fig. 1. Fabrication and characterization of BP/DACHPt. A) Surface coordination of DACHPt and PEGylation on BP. B) DACHPt loading capacities on BP nanosheets (w/w%) with increasing DACHPt/BP feeding ratios. C) TEM image, D) HR-TEM image with inset SAED patterns, E) STEM and EDS mapping (scale bar = 50 nm) of BP/DACHPt. F) Raman spectra of bare BP and BP/DACHPt. G) HR-XPS spectra of P2p and Pt4f.
2.3. In vitro experiments
2.3.1. Stability study of BP formulations
Five BP formulations (bare BP, BP/DACHPt, BP/DACHPtCl2, BP/ CDDP and BP/Pt(NH3)2) in 50 μg/mL were predispersed in air-exposed water under stirring for diﬀerent time periods (0, 12, 24, 72 and 168 h). At each time point, UV–Vis-NIR Vincristine from 300 to 800 nm of the samples was measured. Meanwhile, the photothermal stability of these BP formulations was also tested by monitoring their temperature changes with an infrared thermal imaging camera (Ti450, Fluke, USA) under 808 nm NIR laser irradiation (KS-810F-8000, Shanxi KaiSite Electronic Technology Co., Ltd., China) with power density of 1.0 W/ cm2. The dispersity of BP/DACHPt-PEG in PBS and cell culture media DMEM including 10% FBS at 25 °C was evaluated by DLS.
The DACHPt release from the BP/DACHPt was monitored by the dialysis method. Briefly, 1 mL of BP/DACHPt (1 mg/mL DACHPt) was packaged in a dialysis bag (MWCO = 5 kDa, Shanghai Sangon, China), then incubated in 19 mL of PBS at diﬀerent pH (pH = 7.4 or 5.0). At certain time points, 1 mL of dialyzate was fetched to measure the Pt level by ICP-MS, and 1 mL of fresh medium was then replenished. Photothermal-triggered release of DACHPt studies were performed by irradiating with 808 nm laser (1.0 W/cm2) at pH 5.0 for 10 min.
Meanwhile, the DACHPt released from diﬀerent conditions (original solution, released from pH 7.4 or pH 5.0 after NIR irradiation) were identified by a LC-MS system. The LC-MS system contains a high per-formance liquid chromatographic system (HPLC, Waters Alliance 2996, USA) and a Waters SQ Detector . The separation of DACHPt and other species from the dialysate was performed on a SunFire TW C18
2.3.3. In vitro phototherapeutic eﬀect
Human cervical cancer cell line HeLa were chosen for evaluation of the phototherapeutic eﬀect of BP/DACHPt. Briefly, HeLa cells were incubated in 6-well plates at 37 °C with 5% CO2 for 24 h. Bare PP and BP/DACHPt were predispersed in air-exposed water for 0 or 72 h before test. Afterwards, The HeLa cells were incubated with pretreated bare BP and BP/DACHPt (50 μg/mL) for 4 h and irradiated by the 808 nm laser (1.0 W/cm2) for 10 min. After 30 min incubation, calcein AM (calcein acetoxymethyl ester) and PI (propidium iodide) were used to co-stain the cells to evaluate the photothermal eﬀect of BP/DACHPt by a fluorescence microscope.
To quantify the phototherapeutic eﬀect of BP/DACHPt, the MTT assay was conducted to determine the viability of HeLa cells. Firstly, the cytotoxicity of freshly prepared bare BP was test in four diﬀerent cell lines, including three cancer cell lines and one normal cell line (HeLa, HepG2, A549 and NIH 3T3 cells). Then HeLa cells (5 * 103 cells/well) were seeded into a 96-well plate and incubated at 37 °C overnight. Similarly, bare PP and BP/DACHPt were pretreated in air-exposed water for 0 or 72 h before cell assay. The HeLa cells were incubated with pretreated bare BP and BP/DACHPt (10, 20, 50 μg/mL) for 4 h followed with or without NIR irradiation (808 nm laser at 1.0 W/cm2 for 10 min). After NIR irradiation, the cells were incubated for another 12 h and the cell viability was quantified by MTT using a Bio-Rad 680 microplate reader by formazan absorbance at 490 nm. HepG2 and A549 cells were also used for photothermal eﬃcacy study.
2.3.4. In vitro chemotherapeutic eﬀect
To quantify the chemotherapeutic eﬀect of BP/DACHPt in more detail, the MTT assay of HeLa cells was carried out again. The antic-ancer drug DACHPtCl2 was used as control. HeLa cells (5 * 103 cells/ well) were seeded into a 96-well plate and incubated at 37 °C overnight. The cells were then exposed to DACHPtCl2 and BP/DACHPt solutions with a DACHPt concentration of 0.6, 1.25, 2.5, 5, 10, 20, 40 and 100 μg/mL for another 24, 48 and 72 h. At each time point, the cell viability was quantified by MTT.
Dead Cell Apoptosis Kit [Annexin V-FITC and propidium iodide (PI), Life Technologies] was used to evaluate apoptotic and necrotic cells. HeLa cells (4 * 104 cells/well) were seeded in a 12-well plate and cultured at 37 °C overnight. Cells were treated with blank control and BP/DACHPt (100 μg/mL DACHPt) for 3 h followed with or without ir-radiation (808 nm laser at 1.0 W/cm2 for 10 min). After culture 24 h, the cells were harvested, and the apoptosis kit was used to quantify apoptosis by BD Accuri C6 flow cytometer (USA). Live cells were identified as Annexin V− and PI−, early apoptotic cells as Annexin V+ and PI−, and late apoptotic/necrotic cells as Annexin V+ and PI+ .