Materials
Diethyl ester, N,N-diisopropylethylamine (DIPEA), tri-(2-aminoethyl) amine (TAEA), sodium sulphate anhydrous (Na2SO4), 4-(dimethylamino) pyridine (DMAP), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Darmstadt, Germany). 4-Nitrophenyl chloroformate was obtained from Alfa Aesar (Thermo Fisher, Heysham, Lancashire, UK). Pyridine, anhydrous dichloromethane, triethylamine (TEA), hydrochloric acid (HCl), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and other unlisted chemicals were obtained from J&K Co., Ltd (Beijing, China). Acetonitrile (ACN), methanol, hexene, ethyl acetate, tetrahydrofuran and other solvents were obtained from Oriental Co., Ltd (Hong Kong, China). DSPE-PEG2000 was supplied by Ponsure Biological Co., Ltd (Shanghai, China).
Synthesis of (BODIPY)3-TAEA (BTAEA)
The synthesis of BTAEA was descripted in Additional file 1: Figure S1. Compound 1–3 were synthesized according to the reported methods in our previous study without any modification [20].
Compound 4 (BODIPY-4-NPC): Compound 3 (124 mg, 0.5 mmol) was dissolved in dry tetrahydrofuran (5 mL) in a flask and protected by nitrogen gas in the dark. Then DIPEA (220 µL, 5 eq) was added into the solution. The mixture was cooled to 0 °C and stirred for 10 min. 4-Nitrophenyl chloroformate (4-NPC) (217.7 mg, 4 eq) dissolved in THF was added slowly. Then pyridine (5.5 µL, 0.25 eq) was added. The mixture was stirred for 150 min at room temperature. The organic layer was concentrated under vacuum and the residue was purified with a chromatography column by using 1:1 hexene/DCM (v/v) to give compound 4 as orange powder. The structure and purity of the product were confirmed by 1H-NMR spectroscopy.
Compound 5 (iodine substituted BODIPY-4-NPC): Compound 4 (80 mg, 0.2 mmol) and zinc oxide powder (60 mg, 3.6 eq) were dissolved in dry THF (3 mL) and protected by nitrogen gas in the dark. The mixture was then immersed into ice bath. ICl (100 mg, 3 eq) was dissolved in THF and then added into the mixture drop by drop. The solvent was removed after 15 min of reaction and the residue was purified by silica column eluted by hexene/DCM (1.5:1) to give purple-red powder as the product. The structure and purity of the product were confirmed by 1H-NMR spectroscopy.
Compound 6 (BODIPY3-TAEA, BTAEA): Compound 5 (40 mg, 0.1 mmol) was dissolved in 1.5 mL dry DCM under nitrogen gas and cooled to 0 °C. DIPEA (35 μL, 0.2 mmol) was added and stirred for 15 min. The solution of TAEA (5 μL, 0.03 mmol) in 1 mL dry DCM was slowly added into the above mixture at 0 °C. The mixture was then warmed to room temperature. After stirring for about 1 h, more DIPEA (35 μL) was added. The mixture was continuously stirred for 48 h. Thin-layer chromatography (TLC) was used to confirm the complete consumption of compound 5. DCM/MeOH (20:1) was used to purify the final product with a silica column as purple-red powder. The structure and purity of the product were confirmed by 1H-NMR and ESI–MS spectra.
Fabrication and characterization of BTAEA nanoassemblies
Flash nanoprecipitation method was used to fabricate the nanoassemblies following the reported method [25, 27]. Briefly, BTAEA (10 mg/mL, 5 μL) and DSPE-mPEG2000 (20 mg/mL, 1 μL) were dissolved in DMSO separately and then mixed to form a stock solution. The stock solution was added into 200 μL of filtrated water with vortexing to form BTAEA nanoassemblies (BTNPs). The resulted solution of nanoparticles containing organic solvent can be purified via centrifugation with ultrahigh-speed low-temperature centrifuge (ST 8R, Thermo Fisher Scientific, Waltham, MA, USA). The solution was centrifuged at 3000×g for 10 min, which was repeated for at least three times until no precipitate was observed. The supernatant was then collected and further centrifuged at 30,000×g for 20 min. The nanoparticles were obtained as the precipitate located at the bottom of the tube. The precipitate was resuspended in water or PBS. The concentration of BTAEA was then determined by HPLC. TEM images, mapping images and EDX spectrum was recorded by Philips CM100 transmission electron microscope. Size distribution and zeta potential of the nanoassemblies were measured by dynamic light scattering instrument (ZS90, Malven Instrument, southborough, MA, USA). To test the serum stability of BTNPs, BTNPs were prepared and adjusted to 2 mg/mL in aqueous solution. The BTNPs solution was then diluted by concentrated FBS (2×) to a final concentration of 1 mg/mL. The absorbance at 560 nm was measured at different time points (0 h, 4 h, 12 h and 24 h).
To prepare Pt/BTNPs, PtTPBP was added to the stock solution and the same procedures were used. To prepare PTX/Pt/BTNPs, PtTPBP and PTX were added to the stock solution and the same procedures were used. The concentrations of BTAEA and PTX were determined by HPLC analysis. Loading capacity and encapsulation efficiency were calculated as follow:
$${\mathrm{Loading\,capacity}}\left({\%}\right)=\frac{\mathrm{weight\,of\,the\,payload}}{\mathrm{weight\,of\,the\,nanoparticles }}\times 100{\%}.$$
$${\mathrm{Encapsulation\,efficiency }}\left({\%}\right)=\frac{\mathrm{weight\,of\,the\,loaded\,payload}}{\mathrm{weight\,of\,the\,feeded\,payload}}\times 100{\%}.$$
TD-DFT calculations
Time-dependent density-functional theory (TD-DFT) was used to calculate the energy levels of the S1 states and T1 states of BTAEA and PtTPBP. The calculation procedures were performed based on the Gaussian 16 software package. Geometry optimizations were calculated at the B3LYP/6-31G(d) (LANL2DZ on I) level.
Phosphorescence quenching by TTET
The TTET process was verified by determining the phosphorescence of PtTPBP that can be quenched by BTAEA. The experiments were conducted according to the previous study [20]. Briefly, the phosphorescence of PtTPBP (10–5 M) was recorded in the presence of different concentrations of BTAEA (0, 5 × 10–7 M, 2 × 10–6 M, 4 × 10–6 M, 7 × 10–6 M, and 10–5 M) in the mixed solvent of 90% methanol and 10% toluene. The solution was N2-saturated by purging N2 for 10 min to avoid oxygen quenching. Moreover, the quenching constants (kq) were calculated according to the Stern–Volmer Eq. (1).
$$ \frac{{\text{I}}{0}}{\text{It}}{=}{\text{1+}} \,{\text{k}\small{q}}\left[{\text{Q}}\right],$$
(1)
(I0: phosphorescence intensity of PtTPBP in solution; It: phosphorescence intensity of PtTPBP in the presence of BTAEA; [Q]: concentration of BTAEA).
The rate of TTET process was quantified as kTTET, which can be calculated based on the below Eq. (2).
$${\text{k}{\small{{TTET}}}}{=}\frac{{\text{k}}{\text{q}}}{ \, {\tau}{0}},$$
(2)
(\(\tau\)0: phosphorescence lifetime of PtTPBP without quencher; According to the literature, \(\tau\)0 = 42.92 μs [20]).
Quantitative analysis of BTAEA photolysis
To quantitatively determine the photolysis of BTAEA, the concentrations of BTAEA and released BODIPY were measured by HPLC. The aqueous solution of PtTPBP-loaded BTNPs (Pt/BTNPs) or plain BTNPs (50 µL, 1 mg/mL, on BTAEA basis) was irradiated by 635 nm laser (50 mW/cm2) (LWRL635, Laserwave, Beijing, China) or 530 nm laser (50 mW/cm2) (LWRL530, Laserwave, Beijing, China) for different time periods (0, 1, 3, 5, and 10 min). The irradiated solution was mixed with 50 µL acetonitrile to dissolve the nanoassemblies. The solution was analyzed by HPLC (Angilent 1260 Infinity, CA, USA), and the photolysis profile was recorded based on the irradiation time and the determined concentrations.
Characterization of light-triggered payload release
Nile red-loaded nanoassemblies, NR/BTNPs and NR/Pt/BTNPs, were prepared by the above method. The solutions were put into a cuvette and irradiated by light (635 nm, 50 mW/cm2, 0–10 min). At different time points (0, 1, 2, 3, 5 and 10 min), the fluorescence was measured by a spectrometer (SpectraMax® M4, Molecular Devices LLC, San Jose, CA, USA) without any dilution.
Cell culture
Mouse breast cancer 4T1 cells were purchased from Stem Cell Bank, Chinese Academy of Sciences. Cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 100 units/mL antibiotics (Penicillin–Streptomycin, Gibco) at 37 °C in a 5% CO2 humidified atmosphere.
Cellular uptake analysis
For confocal imaging, 4T1 cells were plated in the confocal plates (Corning, 200350, Cell Culture-Treated) at a density of 5000 cells/well. Different formulations including PBS, NR-labelled BTNPs and Pt/BTNPs (5 μg/mL, on BTAEA basis) were added into the medium. Then the lysosomes were labelled with Lysotracker® Green (Thermo, Heysham, Lancashire, UK). For the light irradiated group, red light (50 mW/cm2) was applied at the bottom of the cell plate for 10 min. After 4 h incubation, the medium was removed and replaced by fresh medium after washing the cells with PBS for 3 times. The cells were observed under a confocal laser scanning microscope (LSM 980, Carl Zeiss, Germany).
Cytotoxicity analysis
Cell viabilities were determined by MTT assay. Briefly, 4T1 cells were cultured on 96-well plates at a primary density of 5000 cells/well and incubated for 24 h before adding the formulations. The medium was replaced with formulation-containing medium at different concentrations. After 4 h incubation, the cells were washed with PBS for 3 times and the light irradiation (635 nm, 50 mW/cm2) was performed. MTT solution (10 μL/well) was added after 24 h of incubation and the OD490, OD570 and OD630 values were measured for the calculation of cell viability. The fluorescence imaging of microtubule was conducted by staining actin with Alexa Fluor 488-phallodin (Thermo Fisher, CA, USA) in 1% BSA-containing PBS. The cells treated with PBS, PTX or PTX/Pt/BTNPs were incubated for 24 h and washed with PBS for 3 times. The CLSM images were collected thereafter.
Intracellular singlet oxygen detection
Singlet oxygen generation was measured in cells by 2′-7′dichlorofluorescin diacetate (DCFH-DA) as an indicator. 4T1 cells were seeded in confocal dishes (Corning, 200350, Cell Culture-Treated) at a density of 5000 cells/well and treated with PBS, BTNPs, Pt/BTNPs or PTX/Pt/BTNPs for 4 h. Subsequently, the cells were washed with PBS for 3 times and incubated with DCFH-DA (10 μM) for 30 min. The cells were irradiated (635 nm, 50 mW/cm2, 10 min) thereafter. The intracellular fluorescence was observed by CLSM imaging to evaluate the intracellular 1O2 generation.
Animals
BALB/c mice (female, 4 weeks, about 15 g) were purchased from the Animal Center of Qinglong Mountain (Nanjing, China). The animal experiments were conducted according to protocol that was approved by the ethics committee of China Pharmaceutical University.
In vivo biodistribution
For the subcutaneous tumor model, mice were injected with 2 × 107 4T1 cells subcutaneously. The mice were then further kept in SPF condition for 5–7 days until tumors were observed and reached at about 100 mm3. Mice were treated with free DiR or DiR/BTNPs via intravenous injection with a dose at 100 μg/kg. Fluorescence imaging was performed at 0, 1, 6, and 24 h post injection. At 24 h, mice were euthanized and the tumors and major organs (heart, liver, spleen, lung, kidney) were excised for ex vivo imaging.
Anti-tumor effects in 4T1 tumor-bearing mice
The antitumor efficacy of the formulations in the presence or absence of light was investigated with 4T1 tumor-bearing mouse model. The mice were randomly divided into 5 groups (n = 4) when the tumor size reached about 100 mm3. Different formulations were intravenously administrated: (1) PBS + hv; (2) PTX; (3) Pt/BTNPs; (4) PTX/Pt/BTNPs; (5) PTX/Pt/BTNPs + hv. For the irradiation groups, light irradiation (635 nm, 200 mW/cm2, 10 min) was performed 24 h post injection. The dose for each treatment was set as 5 mg/kg on PTX basis. The treatments were repeated every three days. Tumor sizes and body weights were measured during the period, and the tumor volume was calculated as V = 1/2 × width2 × length. On Day 14, all of the mice were euthanized, and the tumors and major organs were excised and sliced for H&E staining and immunohistochemical analysis.
Statistical analysis
All experiments were conducted three times or more independently (n ≥ 3). Data were presented as the mean ± standard deviation (SD). The one-way ANOVA-LSD and Independent-Samples t-test were adopted to determine the statistical significance of differences by Graphpad Prism 8.0 software.