Supramolecular combination chemotherapy: a pH-responsive co-encapsulation drug delivery system

Most cancer chemotherapy regimens rely on the use of two or more chemotherapeutic agents. A supramolecular approach that may allow co-delivery of two drugs is described here.


Materials and methods S3
Syntheses and characterization S3 Job plot for interaction of PtC10 with CP6A S6

Materials and Methods
Materials: Carboxylatopillar[6]arene (CP6A) was synthesized according to a literature method. 1 All reagents were purchased commercially and used without further purification unless otherwise noted.
Methods: 1 H NMR spectra were recorded using a JNM-ECA-400 spectrometer.High-resolution mass spectra (HRMS) were recorded on a Bruker Daltonics, Inc. APEXIII 7.0 TESLA FTMS instrument.UV-vis spectra were measured on a UV-2550 ultraviolet-visible spectrophotometer, Shimadzu Research Laboratory Co. Ltd.Fluorescence spectroscopic studies were carried out using a LS-55 fluorescence spectrophotometer, Perkin Elmer Co. Ltd.

Syntheses and Characterization
Figure S1.General synthetic route leading to PtC10.Note: PtC10 is a new compound; it is similar to an analogous C16-carbon chain reported previously. 2

Determination of the association constants.
To assess quantitatively the complexation behavior of these compounds, fluorescence titrations of CP6A with PtC10 were performed at 298 K in phosphate buffer solutions of pH 7.4 and 5.0 per equation 1.We considered that a 1:1 host:guest complex is formed as defined by the association constant (Ka), which satisfied the law of mass action relating the equilibrium concentrations of free host ([H]), free guest ([G]) and hostguest complex ([HG]).The relationship between the total concentration of host ([H]0), guest ([G]0) and their equilibrium concentrations were determined in accord with the law of mass conservation (equatiosn 2-1 and 2-2).Here [H]0 is the initial concentration of the guest (a known parameter that was kept constant in the titration process).Then equations 1 and 2-2 were employed to deduce equation 3. When the fluorescence titration was performed, the intensity of fluorescence (F) corresponds to the combined intensity of the host and the host-guest complex, which were described by molar fractions (equation 4).Both FHG and FH are known parameters in which FH is the fluorescent of [H]0 and FHG is the fluorescent intensity when all host is complexed.

Critical Aggregation Concentrations (CACs) of Free PtC10 and PtC10⊂CP6A
Samples for CAC measurements were prepared by serial dilution from a 96-well plate and transferred into a 384-well black/clear microplate.The absorbance intensity (900 nm) in the samples within the microplate wells were measured using a SpectraMax® M5 plate reader (Molecular Devices, San Jose, CA, USA). 3 The intensity values were then plotted as a function of concentration.The CAC (if any) was determined from the break point in the resulting curve (cf. Figure S9).

Preparation of DOX@PtC10⊂CP6A
Doxorubicin hydrochloride (DOX)-loaded vesicles were prepared as follows: CP6A (3.26 mg, 0.20 mM) was added into double distilled water (9.8 mL) and stirred well.A solution of PtC10 (2.51 mg, 0.40 mM) in ethyl alcohol (0.2 mL) was added dropwise to this solution and the mixture was sonicated for 30 min.Then, DOX (2.32 mg, 0.40 mM) was added into the mixture, which was then stirred overnight.The presumed DOX@PtC10⊂CP6A constructs were purified using a dialysis bag (MW: 8000 -14000) placed in distilled water, with the procedure being repeated several times until free DOX in the outside solution could no longer be detected by HPLC.This whole purification process was performed in the absence of light.being stable under these experimental conditions (mean ± SD, n = 3).
The drug encapsulation efficiency was calculated as follows: Where W0 is the mass of drug added, Wt is the mass of drug encapsulated in vesicles.The mass of DOX and PtC10 were measured by HPLC and calculated relative to a standard calibration curve over the 0.078 to 40.00 µM concentration range in double distilled water.By diluting five-fold with 1% Triton X-100 and twofold with ethyl alcohol, nearly 100% release of DOX and PtC10 from the drug-loaded vesicles could be achieved.

In Vitro Drug Release of DOX@PtC10⊂CP6A
The release of DOX and PtC10 in vitro was tested using dialysis bags (molecular weight cutoff: 8000 -14000).Phosphate buffer solution (0.1 mM) with different pH (5.0 and 7.4) was used as release medium.Free DOX or DOX@PtC10⊂CP6A (1 mL of a corresponding aqueous solutions) was added to the dialysis bag and immersed in 10 mL of the release medium under stirring (200 rpm, preheated to 37 ± 1 °C). 1 mL dialysate samples were withdrawn and replaced with an equal volume of fresh media at predetermined intervals.The concentration of DOX in the medium was determined by HPLC.The cumulative amount of DOX released was calculated as follows: Where m0 is the total mass of drug in free DOX solution or DOX@PtC10⊂CP6A, V0 is the initial medium volume, Vs is the sampling volume, C0 and Ct are the drug concentrations, t and n are the sampling times.All assays were performed in triplicate in the absence of light.

In Vitro Cytotoxicity Studies
The relative cytotoxicity of CP6A against two cell lines was assessed in vitro using CCK-8 according to the manufacturer's instructions.Human liver hepatocellular carcinoma cells (HepG-2) were seeded into 96-well plates at a density of 8000 cells/well in 100 µL of MEM supplemented with 10% FBS, 1% penicillin, and 1% streptomycin.Likewise, human normal liver cells (LO2) were seeded into 96-well plates at a density of 8000 cells/well in 100 µL of complete DMEM supplemented with 10% FBS, 1% penicillin, and 1% streptomycin and cultured for 24 h in 5% CO2 at 37 °C.CP6A was dissolved in PBS and then diluted to the required concentration.It was then added to the cell-containing wells which were further incubated at 37 °C under 5% CO2 for 72 h.Subsequently, 10 µL of CCK-8 was added into each well and incubated for another 0.5 h.The plates were then measured at 450 nm using a SpectraMax® M5 plate reader (Molecular Devices, San Jose, CA, USA).All experiments were carried out five independent times.Cell viability was calculated as follows: Cell Viability = OD abca − OD defgh OD ijgakje − OD defgh × 100% Where ODblank is the optical density of blank well (medium and CCK-8 reagent), ODtest is the optical density of the test group and ODcontrol is the optical density of the control group., where D1 and D2 represent the concentration values of drug 1 and drug 2 when used in combination to inhibit half the cell growth, respectively, and Dm1 and Dm2 represent the half-maximum inhibitory concentration values of each drug alone.
The cells were then treated with several internalization inhibitors for 0.

In Vivo Antitumor Efficacy
Six-week-old male BALB/c nude mice (~20 g body weight) were purchased from the Beijing Experimental Animal Center (Beijing, China).A total of 1 × 10 6 HepG-2 cells in 200 µL of saline were inoculated subcutaneously into the right dorsal flanks of the mice.The mice were normally fed after inoculation for 1 week.total, 30 mice were randomly divided into five groups.The control group (5% glucose to mimic what is used in the clinic for OX administration) and the other groups containing 5% glucose and either OX (8.90 mg•kg -1 ), DOX group (4.00 mg•kg -1 ), OX+DOX group (8.90 mg•kg -1 OX + 4.00 mg•kg -1 DOX, molar ratio = 3.25) and DOX@PtC10⊂CP6A group (18.29 mg•kg -1 CP6A + 14.06 mg•kg -1 PtC10 + 4.00 mg•kg -1 DOX) were subject to intravenous (i.v.) injection in mice at different time points (on days 1, 3, 5, 7, and 9).After treatment, the tumor volumes and the weights of the mice were recorded at different time points.On day 11, the mice were sacrificed and the tumors were separated from the animals and weighed.All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the AAALAC, and were approved by the Animal Care and Use Committee of the National Beijing Center for Drug Safety Evaluation and Research.Best efforts were made to minimize the number of animals used and their suffering.Tumor volumes were calculated as follows: Tumor Volume ( s ) = 1 2 ×  ×  9 In this equation, a and b represent the largest and smallest tumor diameters, respectively.

Figure S7 .
Figure S7.Job's plot for the interaction of CP6A with PtC10.The maximum at a mole fraction is consistent with a proposed 1:1 stoichiometry for the complex.To construct the plot, the absorbance at 290 nm was monitored as a function of mole fraction.The study was conducted at pH 5.0 in PBS; [CP6A] + [PtC10] = 1.0 × 10 -5 M.

Figure S16 .
Figure S16.Diameter of PtC10⊂CP6A and DOX@PtC10⊂CP6A before (0 h) and after being incubated in double-distilled water for the indicated time periods as determined by DLS.No significant size change occurred over the course of 72 h, a finding interpreted in terms of PtC10⊂CP6A and DOX@PtC10⊂CP6A

Figure S17 .
Figure S17.Calibration curves obtained by HPLC and used for calculating (a) the DOX concentration and (b) the PtC10 concentration in the release studies described in the main text.

Figure S19 .
Figure S19.Relative cell viabilities of (a) HepG-2 and (b) LO2 cells after incubation for 72 h with CP6A at the indicated concentrations.Cell death was then measured by using a CCK-8 assay (mean ± SD, n = 5).
5 h: 0.1 M 5-(N-ethyl-N-isopropyl) amiloride (EIPA) for inhibiting macropinocytosis; 0.4 M sucrose, and 0.05 M chlorpromazine (CP) for clathrin-coated vesicle formation; 0.05 M ammonium chloride (AC) for lysosome function.Cells were also incubated at 4 °C to minimize all energy-dependent uptake pathways.The cells were then rinsed with PBS before DOX@PtC10⊂CP6A was added and the cells incubated for 4 h.After 4 h, the cells were harvested and washed three times with cold PBS and resuspended in 500 µL PBS.DOX fluorescence was detected by flow cytometery.Cells incubated without inhibitor were used as a negative control.