Cationic pillar[6]arene/ATP host–guest recognition: selectivity, inhibition of ATP hydrolysis, and application in multidrug resistance treatment

The efficacy of DOX·HCl against drug resistant human breast cancer cells is enhanced in the presence of a water-soluble pillar[6]arene (WP6) due to the inhibition of ATP hydrolysis upon formation of WP6⊃ATP in cells, providing a novel strategy to overcome multidrug resistance.

Solvents were either employed as purchased or dried according to procedures described in the literature. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance Ⅲ-400 spectrometry. 2D NOESY NMR spectrum was collected on a Bruker Avance DMX-500 spectrometer with internal standard TMS. Low-resolution electrospray ionization (LRESI) mass spectra were obtained on a Bruker Esquire 3000 plus mass spectrometer

Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS)
Studies. The morphologies of DOX·HCl loaded WP6/FA-PEG-b-PAA ternary PIC micelles were revealed by TEM. The ternary micelles (the concentration of the polymer was 0.75 mg mL -1 ) were prepared first in water. TEM samples were prepared by drop- S2 coating the solution on a carbon-coated copper grid. TEM experiments were performed on a HT-7700 instrument. The corresponding solution was left to stand overnight and the insoluble precipitate was eliminated by using a microporous membrane before being used for DLS tests. Dynamic light scattering (DLS) measurements were carried out using a 200 mW polarized laser source Nd:YAG (λ = 532 nm). The polarized scattered light was collected at 90° in a self-beating mode with a Hamamatsu R942/02 photomultiplier. The signals were sent to a Malvern 4700 submicrometer particle analyzer system. Cell Culture. A549, KB and MCF-7/ADR cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells grew as a monolayer and were detached upon confluence using trypsin (0.5% w/v in PBS). The cells were harvested from cell culture medium by incubating in the trypsin solution for 5 min. The cells were centrifuged, and the supernatant was discarded. A 3.00 mL portion of serum-supplemented DMEM was added to neutralize any residual trypsin. The cells were resuspended in serum-supplemented DMEM at a concentration of 1.00 × 10 4 cells/mL. Cells were cultured at 37 °C and 5% CO 2 .

In Vitro Cell Accumulation of the Ternary PIC Micelles Determined by Flow
Cytometry. Cellular uptake of DOX·HCl-loaded WP6/FA-PEG-b-PAA ternary PIC micelles was measured by flow cytometry. A549 and KB cells were seeded at a density of 3.00 × 10 5 cells/well in 12-well cell culture plates. The cells were left to grow for 24 h in DMEM media containing 10% FBS at 37 o C in 5% CO 2 atmosphere. After 24 h, DOX·HCl (5.00 μg/mL) loaded WP6/FA-PEG-b-PAA ternary PIC micelles were added to the wells and the cells were incubated for 30 min, 1 h, 2 h, 3 h, and 4 h, respectively.
Following incubation, cells were rinsed twice with PBS to remove residual ternary PIC micelles. Cells were harvested by trypsinization and resuspended in 500 μL of PBS for flow cytometry analysis using the FACS Calibur flow cytometer (BD Facsealibur). Data shown are the mean fluorescent signal for 1.00 × 10 4 cells. Cells that were not treated with the ternary PIC micelles were used as a control. Data was analyzed using the FlowJo software. MTT solution was removed, and the insoluble formazan crystals that formed were dissolved in 100 μL of dimethylsulfoxide. The absorbance of the formazan product was measured at 570 nm using a spectrophotometer (Bio-Rad Model 680). Untreated cells in media were used as a control. All experiments were carried out with four replicates.

Synthesis of 2:
To a solution of 1 (16.1 g, 50.0 mmol) in chloroform (300 mL), paraformaldehyde (3.00 g, 100 mmol) was added under nitrogen atmosphere. Then boron trifluoride diethyl etherate (10 mL) was added to the solution and the mixture was stirred at room temperature for 3 h. Water (50 mL) was added to quench the reaction. The mixture was filtered and the solvent was removed. The residue was dissolved in dichloromethane. The organic layer was dried over anhydrous Na 2 SO 4 and evaporated to afford the crude product, which was isolated by flash column chromatography using ethyl acetate/petroleum ether (1:5) to give 2 as a white solid (2.10 g, 12%), mp: 81.682.9 C.

Synthesis of N 3 -PEG-OH:
A mixture of TsO-PEG-OH (6.64 g, 1.00 mmol) and NaN 3 (1.63 g, 25.0 mmol) was heated in N,N'-dimethylformamide (30.0 mL) at 90 °C for 12 h. The reaction mixture was diluted with dichloromethane (500 mL) and washed with water (3 × 200 mL). The organic phase was dried over magnesium sulfate and filtered.
The solvent was removed under reduced pressure to afford N 3 -PEG-OH as a white solid (5.09 g, 78%). Molecular weights and compositions of N 3 -PEG-OH were determined by GPC (Fig. S7) and 1 H NMR spectroscopy (Fig. S8b). Synthesis of N 3 -PEG-Br: Triethylamine (2.02 g, 20.0 mmol) was added dropwisely to a solution of 2 (3.22 g, 0.500 mmol) and α-bromoisobutyryl bromide (1.15 g, 5.00 mmol) in dry THF (50 mL) at 0 °C. After warming up to room temperature, the mixture was further stirred for 12 h. After removal of insoluble salts by filtration, the solvent was removed on a rotary evaporator. The residues were dissolved in THF and precipitated into an excess of cold diethyl ether. The above dissolution-precipitation cycle was repeated three times. The final product was dried in a vacuum oven overnight at room temperature, yielding a white viscous solid (2.83 g, yield: 86%). Molecular weights and compositions of N 3 -PEG-Br were determined by GPC (Fig. S7) and 1 H NMR spectroscopy (Fig. S8c).          T∆S° > 0; |∆H°| < |T∆S°|), which indicated that the complexations were driven by entropy changes. The reason is that the "high-energy" water molecules encapsulated in the cavity of WP5 (or WP6) are released to the aqueous bulk by the formation of inclusion hosteguest complexes, which are favorable for entropic gain. S2

Fig. S25
Fluorescence spectra of WP6 (2.00 × 10 -6 M) in a phosphate buffer solution at room temperature with different concentrations of ATP from 0 to 5 equiv.