A tumour-selective cascade activatable self-detained system for drug delivery and cancer imaging

Achieving the activation of drugs within cellular systems may provide targeted therapies. Here we construct a tumour-selective cascade activatable self-detained system (TCASS) and incorporate imaging probes and therapeutics. We show in different mouse models that the TCASS system accumulates in solid tumours. The molecules show enhanced accumulation in tumour regions via the effect of recognition induced self-assembly. Analysis of the molecular penetration in tumour tissue shows that in vivo self-assembly increases the penetration capability compared to typical soft or hard nanomaterials. Importantly, the in vivo self-assembled molecules exhibit a comparable clearance pathway to that of small molecules, which are excreted from organs of the reticuloendothelial system (liver and kidney), while are relatively slowly eliminated from tumour tissues. Finally, this system, combined with the NIR probe, shows high specificity and sensitivity for detecting bladder cancer in isolated intact patient bladders.

After further 6 h of incubation, the cells were washed twice by PBS and stained with bisBenzimide H 33342 trihydrochloride (Hoechst 33342) (1 mg ml -1 ; Life Technologies) at 37 °C for 10 min. Finally, the cells were washed by PBS twice at 4 °C and immediately observed under confocal laser scanning microscopy (UltraVIEW VoX).
Cyanine dye (Cy) synthesis. Cyanine dye synthesis method reference to previously published work 1 .
TEM sample preparation. Samples of the supramolecular aggregates were prepared by dropping a solution of molecules P1 and 5 in 1% HFIP into aqueous solutions. The mixtures were vortexed and set aside for 24 h before analysis. Transmission electron microscope (TEM) studies were carried out on a Tecnai G2 20 electron microscope operating at an accelerating voltage of 200 keV. The TEM samples were prepared by placing nanofibrils droplets on copper grids for 5 min and removing the excess droplets. Finally, the samples were stained by uranyl acetate for 2 min, then washed with water.
The photostability of Cyanine dye. Based on the mechanism of photoreaction, the equation of photoreaction is shown as below 2 : As a constant, the concentration of O2 in the solution full of oxygen is about 3×10 − 4 mol L -2 . So the formulas are as follows: [Cy]0 is the initial concentration of the molecule 1 or 4, [Cy]t is the concentration of the molecule 1 or 5 after the irradiation of t min. Based on Beer rule, it has the linear relation between the concentration of the molecule 1 or 4 and the absorbance in maximal wavelength for the low concentration cyanine dye. So the formula is as follows: A0 is the absorbance in maximal wavelength before the irradiation, and At is the absorbance in maximal wavelength after the irradiation. The slope of the line, k, is the speed constant of the photodegradation reaction.
The pathway of molecule 1 entering cells. First, specific endocytic inhibitors were used to explore uptake pathways of molecule 1. Filipin Ⅲ (5 μg/ml), chlorpromazine hydrochloride (10 μg/ml) or 5-(N, Ndimethyl) amiloride hydrochloride (10 μM) were added to H460 cells in serum-free culture medium for 1 h, at 37℃. The untreated H460 cells were used as control group. Subsequently, H460 cells were treated with molecule 1 (50 uM), after 2 h of incubation at 37 ℃ the medium was removed and the cells were washed three times with PBS and analysed by confocal laser scanning microscopy (UltraVIEW VoX).
Detection of caspase-3 activity. H460 cells and 293T cells were treated with P7 (50 μM) for 1 h. In the other group, the H460 cells were firstly treated with P6, then treated with caspase-3 inhibitor zVDK-FMK (10 μM). After treated with corresponding molecule, each group H460 cells were trypsinized and collected into 1640 cell culture media. The cells were collected by centrifugation at 600 g, 4 ℃ for 5 minutes, and the supernatant was carefully aspirated and washed once with PBS.
After that, cell lysis buffer (approximately 2-10 million cells/ml) was added. After resuspended and lysed in an ice bath at 1℃, 16000g for 15 minutes, the supernatant was transferred to a pre-cooled centrifuge tube in an ice bath. For each group, 20 μM (final concentration) Ac-DEVD-AMC and 100μl cell lysate were added to 1 ml Protease Assay Buffer [20 mM HEPES (pH 7.5), 10% glycerol, 2 mM DTT]. The reaction mixtures were incubated for 1 hour at 37 °C. The AMC released from Ac-DEVD-AMC was measured using a spectrofluorometer with an excitation wavelength of 380 nm and an emission wavelength range of 400-550 nm.
Flow cytometry analysis. A density of 1 × 10 5 H460 cells per well were seeded in the 6-well plates in 1640 medium which contains 10% fetal bovine serum and 1% penicillin-streptomycin, then cultured at 37 °C in a humidified atmosphere with 5% CO2 for 16 h. Then molecules 1, 2 and 3 (50 μM) were added to each well, and the cells were incubated for additional 6 h, 12 h and 24 h, respectively. The apoptosis of the collected cells stained by Annexin V-FITC and PI (propidium iodide) was studied by flow cytometry.
Cell viability assays. CCK-8 (cell counting kit-8) assay was carried out to investigate the cell viability.
H460 cells were utilized to evaluate the cytotoxicity of molecules 1, 2 and 3 by the CCK-8 assay.   8.7, 11.2, 14.0, 17.5, 35.0 (μg/mL), respectively. The control group represented PBS buffer. b. Fluorescence intensity (relative to the background signal) increased linearly with the concentration (R 2 = 0.99). c. The Pep-nanofibers (14 mg/kg, n = 3) on H460-tumour-bearing mice after intravenous administration in three groups. The mice were sacrificed at 48 h post injection. All major organs (i.e., heart, liver, spleen, lung, kidney) and tumour were collected, weighed, and homogenized (0.3 mL of lysis buffer per 100 mg of tissue). The homogenous tissue solutions were obtained for fluorescence imaging.
Supplementary Figure 48| The cytotoxicity assay： tumour cells (H460) were treated with the molecule 1-DOX (100 nM), free DOX (100 nM) and the molecule 3-DOX (100 nM). The sample solutions were added to each well, and the cells were incubated for additional 24 h. Then each well was added with 10 μL of CCK-8 solutions and cultured for another 4 h. Microplate reader was used to measure the UV-Vis absorptions of sample wells (Asample), Ablack and control wells (Acontrol) at a test wavelength of 450 nm and a reference wavelength of 690 nm, respectively. Cell viability (%) was equal to (Asample-Ablank)/ (Acontrol-Ablank) x 100. All the experiments were performed in quadruplicate. Data are presented as the mean ± s.d. (n = 4). *** p < 0.001, p values were performed with one-way ANOVA followed by post hoc Tukey's test for the indicated comparison. n. s. represents no significance.

Supplementary Figure 50|
The fluorescence images of the ex-bladder in orthotopic bladder tumour mice. The mice were treated with 100 uL molecule 1 (50 μM) in PBS for 1 h through intravesical instillation. Histology evaluations of the tissues were collected from the signal site in the ex-bladder.

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Supplementary Figure 51| The S/N ratio of NIR fluorescence in tumour and its surrounding tissues in Fig.6b.