br In vivo experiments br
2.4. In vivo experiments
2.4.1. Tumor model establishment
Female sever combined immunodeficient (SCID) mice (4–5 weeks old) were purchased from the Sun Yat-sen University Laboratory Animal Center and fed in an SPF (specific pathogen free) class experi-mental animal room. The protocols for in vivo experiments were agreed by the Administrative Committee on Animal Research in Sun Yat-sen University. 100 μL of HeLa cells (about 5 * 106 cells) was sub-cutaneously injected into the right hip of each mouse to establish the Chemical Engineering Journal 375 (2019) 121917
HeLa cells xenograft model. The volume of tumor (V) was calculated by equation: V = a * b2/2, where a and b respectively represents the length and width of the tumor. Relative tumor volume was calculated as V/V0, where V0 represents the initial volume of tumor before treatment. The treatments began when the tumor volume reached 100 mm3.
2.4.2. In vivo antitumor eﬃcacy via intratumoral injection
Firstly, to acquire the photothermal images, HeLa tumor-bearing mice were randomly divided into four groups (1–4, n = 5) and in-tratumorally (i.t.) injected with 20 μL of saline, bare BP, BP/DACHPt and BP/DACHPt (0.5 mg/mL DACHPt and corresponding BP dose). Then the mice in 1, 2 and 4 groups were anesthetized and the tumor sites were irradiated with a 808 nm NIR laser at 1.5 W/cm2 for 10 min. Then the antitumor activity was evaluated in accordance with relative tumor volume V/V0 every 2 days until the mice were sacrificed at day 14.
2.4.3. Pharmacokinetics analysis
To analyze the in vivo pharmacokinetics profile of BP/DACHPt-PEG, three SCID mice were intravenously (i.v.) injected with BP/DACHPt-PEG (6 mg DACHPt/kg body weight) via tail vein. At defined time periods (0.5, 1, 2, 4, 6, 12 and 24 h), 20 μL of blood was collected from orbital cavity, heparinized, and centrifuged (15000 rpm, 10 min) to obtain the plasma . The plasma samples were decomposed by heating in nitric KIN59 and their Pt contents were quantified by ICP-MS.
2.4.4. Biodistribution analysis
To evaluate the in vivo biodistribution of BP/DACHPt-PEG, HeLa tumor-bearing mice were randomly divided into three groups (n = 3) and injected with BP/DACHPt-PEG (6 mg DACHPt/kg body weight) via tail vein. 2, 12 and 24 h after injection, the mice was sacrificed and the major organs including heart, liver, spleen, lung, kidney and tumor were collected. After weighed, all organs were suspended in 1.0–1.5 mL of DI water, homogenized and centrifuged (15000 rpm, 10 min) to ob-tain the supernatant. After decomposed similarly, the Pt contents of the supernatant were also quantified by ICP-MS.
2.4.5. In vivo antitumor eﬃcacy via intravenous injection
HeLa tumor-bearing mice were randomly divided into four groups (n = 5) treated with Saline (1), NIR (2), BP/DACHPt-PEG (3), or BP/ DACHPt-PEG + NIR (4) (6 mg/kg body weight on a Pt basis) via tail vein injection. During the irradiation, an infrared thermal image camera was used to monitor the temperature changes and infrared thermographic maps. Tumor volumes were then measured as well as body weight every 2 days until the mice were sacrificed at day 14.
2.4.6. Histopathology study
The tissues were treated with hematoxylin and eosin (H&E) and then their histopathology was assessed by optical microscopy . At day 14, the mice were sacrificed and the major organs including heart, liver, spleen, lung, kidney and tumor were collected, fixed with 10% formalin, embedded with paraﬃn and sliced into 5 μm sections. After staining with H&E, the slices of tissues were observed by Nikon Eclipse 600 microscope (Melville, NY, USA).
2.5. Statistical analysis
All the experiments were repeated at least three times unless otherwise stated. The results are presented as means ± s.d., and the significance of diﬀerence observed between study groups was analyzed
Fig. 2. Enhanced stability and drug release profiles of BP/DACHPt. Absorption spectra of A) bare BP and B) BP/DACHPt, C) variation of the absorption ratios at 440 nm (A/A0) of BP and BP/DACHPt, photothermal heating curves of D) bare BP, E) BP/DACHPt and F) BP/DACHPtCl2 dispersed in air-exposed water for 0, 12, 24, 72 and 168 h. The 808 nm laser was used as the irradiation source. Selected AFM scans of G) bare BP and H) BP/DACHPt sheets after exposure to the humid air for 24 h. I) Drug release profiles of BP/DACHPt at pH 7.4 and pH 5.0 (in the absence or presence of NIR irradiation), ↓: NIR irradiation for 10 min.
3. Results and discussion
3.1. Morphology and characterization
According to the previous literature, black phosphorus (BP) na-nosheets with the size of approximately 100 nm were obtained through liquid exfoliation of bulk BP crystals in NMP . Meanwhile, Cl atoms were eliminated from antitumor agent dichloro(1,2-diaminocyclo-hexane) platinum (II) (DACHPtCl2) into DACHPt, the active species of oxaliplatin . To verify the coordination between BP nanosheets and DACHPt, BP/DACHPt complexes with diﬀerent DACHPt/BP feeding ratios (0.5, 1, 2, 3, 4, 5, 6 and 8) were detected by ICP-MS to measure the drug loading capacity. Fig. 1B showed that with increasing DACHPt/BP feeding ratio, the loading capacity of DACHPt almost lin-early rose and reached maximum at the ratio of 4. The maximum loading capacity was about 202% which significantly exceeded those of routine nanocarrier systems (10–30%) [44,46,49,50]. Considering that the BP/DACHPt complex was prepared from organic solvent but not water, they indeed interacted strongly through coordination instead of hydrophobic and other van der Waals interactions. Then the complex structure was further characterized. Transmission electron microscopy (TEM) and atomic force microscope (AFM) showes that BP/DACHPt had a nanosheet structure (Fig. 1C and S2) with the size and thickness