Biodegradable black phosphorus-based nanospheres for in vivo photothermal cancer therapy

Abstract

Photothermal therapy (PTT) offers many advantages such as high efficiency and minimal invasiveness, but clinical adoption of PTT nanoagents have been stifled by unresolved concerns such as the biodegradability as well as long-term toxicity. Herein, poly (lactic-co-glycolic acid) (PLGA) loaded with black phosphorus quantum dots (BPQDs) is processed by an emulsion method to produce biodegradable BPQDs/PLGA nanospheres. The hydrophobic PLGA not only isolates the interior BPQDs from oxygen and water to enhance the photothermal stability, but also control the degradation rate of the BPQDs. The in vitro and in vivo experiments demonstrate that the BPQDs/PLGA nanospheres have inappreciable toxicity and good biocompatibility, and possess excellent PTT efficiency and tumour targeting ability as evidenced by highly efficient tumour ablation under near infrared (NIR) laser illumination. These BP-based nanospheres combine biodegradability and biocompatibility with high PTT efficiency, thus promising high clinical potential.

Figure 1. Morphology and characterization.

(a) TEM (scale bar, 20 nm) and (b) high-resolution TEM images of the BPQDs (scale bar, 1 nm). (c) Statistical analysis of the size of 200 BPQDs based on the TEM images. (d) AFM image of the BPQDs (scale bar, 200 nm). (e) Height profiles along the white lines in d. (f,g) SEM images of the BPQDs/PLGA NSs (scale bar, f: 1 μm; g: 100 nm). (h) Statistical analysis of the size of 200 BPQDs/PLGA NSs according to the SEM images. (i) TEM image of the BPQDs/PLGA NSs (scale bar, 200 nm) with the inset displaying the magnified TEM image of a BPQDs/PLGA NS. (j) Energy dispersive X-ray spectroscopy analysis of the BPQDs/PLGA NSs.

Figure 2. Stability evaluation under ambient conditions.

(a) Photographs and (b,c) Absorption spectra of the BPQDs and BPQDs/PLGA NSs with the same amount of BPQDs (20 p.p.m.) after storing in water for different periods of time. Insets in (b,c): tyndall effect and variation of the absorption ratios (A/A₀) at 808 nm. (d,e) Raman scattering spectra acquired from the BPQDs and BPQDs/PLGA NSs, respectively, after storing in water for 0 and 8 days. (f,g) Photothermal heating curves of the BPQDs and BPQDs/PLGA NSs, respectively, after storing in water for different periods of time and being irradiated with the 808 nm laser (1 W cm⁻²) for 10 min.

Figure 3. Biodegradation performance.

(a) Absorbance spectra of the BPQDs/PLGA NSs (internal BPQDs concentration is 10 p.p.m.) dispersed in PBS for 0 h, 24 h and 8 weeks with the inset showing the corresponding photographs. (b) Photothermal heating curves of the BPQDs/PLGA NSs dispersed in PBS for 0 h, 24 h and 8 weeks and irradiated with the 808 nm laser (1 W cm⁻²) for 10 min. (c) Residual weight of the BPQDs/PLGA NSs after degradation in PBS as a function of time (n=5; ^*P<0.05, ^**P<0.01, ^***P<0.001; ANOVA). (d) SEM images (scale bars, 500 nm) of the BPQDs/PLGA NSs after degradation in PBS for 1, 4 and 8 weeks together with the corresponding TEM image (scale bar, 200 nm) of the NSs after degradation for 8 weeks. (e) Schematic representation of the degradation process of the BPQDs/PLGA NSs in the physiological environment.

Figure 4. Cell experiments.

(a) Relative viability of the human skin fibroblast normal cells, MCF7 cancer cells and B16 melanoma cells after incubation with BPQDs/PLGA NSs (internal BPQDs concentrations of 0, 2, 5, 10, 20, 50 and 100 p.p.m.) for 48 h. (b) Relative viability of the MCF7 and B16 cells after incubation with BPQDs/PLGA NSs (internal BPQDs concentrations of 0, 2, 5, 10 and 20 p.p.m.) for 4 h after irradiation with the 808 nm laser (1 W cm⁻²) for 10 min. (c) Corresponding fluorescence images (scale bars, 100 μm for all panels) of the cells stained with calcein AM (live cells, green fluorescence) and PI (dead cells, red fluorescence).

Figure 5. In vivo toxicity.

(a) Haematological data of the mice intravenously injected with the BPQDs/PLGA NSs at 1, 7 and 28 days post-injection. The terms are following: white blood cells, red blood cells, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets and haematocrit. (b) Blood biochemical analysis of the NSs-treated mice at 1, 7 and 28 days post-injection. The results show the mean and s.d. of aminotransferase, aminotransferase, total protein, globulin, total bilirubin, blood urea nitrogen, creatinine and albumin (AL B). (c) Histological data (haematoxylin and eosin stained images) obtained from the liver, spleen, kidney, heart and lung of the NSs-treated mice at 1, 7 and 28 days post-injection (scale bars, 100 μm for all panels).

Figure 6. Pharmacokinetic and biodistribution analysis.

(a) Blood circulation curve of the Cy5.5-labelled BPQDs/PLGA NSs determined by measuring the Cy5.5 fluorescence intensity in the blood of the MCF7 tumour-bearing Balb/c mice at different time points post-injection of the NSs. The pharmacokinetics obeys a typical two compartment model (as shown by the fitted curve). (b) In vivo fluorescence images of the NSs-treated mice at different time points post-injection. (c) Ex vivo fluorescence images of the tumour and major organs from the NSs-treated mice at 24 h post-injection. H, heart; I, intestine; K, kidney; Lu, lung; Li, liver; Sp, spleen; St, stomach; T, tumour. (d) Fluorescence microscopy images of the tumour sections at macro-organizational level (scale bars, 1 mm) and micro-organizational level (scale bars, 50 μm) from the NSs-treated mice. The NSs are shown in red and the nuclei are shown in blue by staining with DAPI. (e) Quantitative biodistribution analysis of the NSs in mice by measuring the Cy5.5 fluorescence intensity in the tumours and major organs at different time points post-injection.

Figure 7. In vivo photothermal cancer therapy.

(a) Infrared thermographic maps and (b) Time-dependent temperature increase in the MCF7 breast tumour-bearing nude mice irradiated by the 808 nm laser (1 W cm⁻²) at 24 h after separate intravenous injection with 100 μl of PBS, PLGA NSs, BPQDs (1 mg ml⁻¹) and BPQDs/PLGA NSs (1 mg BP ml⁻¹) with the colour bar referring to the relative temperature. (c) Growth curves of MCF7 breast tumour in different groups of nude mice treated with PBS, PLGA NSs, BPQDs (1 mg ml⁻¹) and BPQDs/PLGA NSs (1 mg BP ml⁻¹) with the NIR laser irradiation.