br An aqueous phase consisting of
An aqueous phase consisting of 0.1 M HCl (20 ml) containing Pluronic F68 (2 mM, Sigma, USA) and Kolliphor HS15 (6 mM, Sigma, Germany) was added to the oil phase and immediately sonicated for 3 min on ice (6 × 30 sec intervals, 60% amplitude, Branson Ultrasonics digital sonifier 450, USA). The solution was rotated (15 rpm, SB3 ro-tator, Stuart, UK) at room temperature overnight before adjusting the pH to 5 using 1 M NaOH. The polymerization was continued for 5 h at room temperature on rotation. The dispersion was dialyzed (Spectra/ Por dialysis membrane MWCO 100,000 Da, Spectrum Labs, USA) against 1 mM HCl to remove unreacted PEG. The size, polydispersity index (PDI) and the zeta potential of the NPs were measured by dy-namic light scattering and laser Doppler Micro-electrophoresis using a Zetasizer Nano ZS (Malvern Instruments, UK). To calculate the amount of encapsulated drug, the drug was extracted from the particles by dissolving them in acetone (1:10), and quantified by liquid chromato-graphy coupled to mass spectrometry (LC-MS/MS) as described below.
2.2. CBZ quantification by LC-MS/MS
CBZ, as the pure chemical or part of NPs, was quantified by LC-MS/ MS, using an Agilent 1290 HPLC system coupled to an Agilent 6490 triple quadrupole mass spectrometer. The HPLC column was an Ascentis Express C8, 75 × 2.1 mm, 2.7 μm particles size with a 5 × 2.1 mm guard column of the same material (Sigma), run at 40 °C. Eluent A was 25 mM formic 14605-22-2 in water and eluent B was 100% methanol, and flow rate was 0.5 ml/min. The mobile phase gradient was isocratic at 55% B for 1.5 min, then from 55% to 80% B over 1 min, followed by 1 min washout time and subsequently column re-equili-bration. Injection volume was 5.00 μl. MS detection was in positive ESI mode (Agilent Jetstream) quantified in multiple reaction monitoring (MRM) mode using the transition m/z 858.3 → 577.2. The parent ion was chosen to be the Na adduct as this gave the best sensitivity. Similarly, the hexadeuterated internal standard was detected on the 864.4 → 583.2 transition. Both analytes were run at 380 V fragmentor and 20 V collision energy.
Reference standards were used for accurate quantification. The unlabeled CBZ standard was the same as used for synthesis (see above) at > 98% purity. Hexadeuterated CBZ internal standard was purchased from Toronto Research Chemicals (Toronto, Canada; catalogue number C046502 at 99.6% isotopic purity). Standards were dissolved in acetone and were used to build an unlabeled standard series spanning at least five concentration points
The limit of quantification (LOQ) was calculated from six replicate quantifications of the lowest concentration point in the standard curves (0.1 ng/ml), specifically as the average plus six standard deviations; this amounted to an LOQ of 0.19 ng/ml (signal/noise ratio ˃20). Accuracy based on the same standard sample set was 8.8% and precision was 18.0%.
2.3. Processing of tissue samples before LC-MS/MS analyses
In order to process the tissue samples such that their CBZ content could be quantified, we developed a protocol for enzymatic digestion of tissue followed by extraction and quantification of CBZ using the LC-MS/MS method described above. The enzyme buffer consisted of Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, USA, 41965039) with 1% (v/v) penicillin-streptomycin stock solution (Sigma-Aldrich, P0781) to a final concentration of 100 U/ml penicillin and 100 μg/ml streptomycin, 0.125 mg/ml papain (Merck, F275644), 2.5 mg/ml trypsin (Sigma-Aldrich, T7409), 0.8 mg/ml collagenase (Sigma-Aldrich, C7926), 0.69 mg/ml hyaluronidase (Sigma-Aldrich, H3506) and 1% (v/v) Triton X-100 (Sigma-Aldrich, T-8787). To de-termine the biodistribution of CBZ, frozen organs (liver, spleen, lymph nodes, kidneys, tumors) were thawed and freshly prepared enzyme buffer was added at 1 ml per 50 mg tissue; the entire organs were di-gested. The samples were heated to 37 °C for 72 h with vortexing once a day, until the tissue was completely dissolved. Stability of CBZ under these conditions was verified by incubation of known concentrations of the free drug. The tissue digests, as well as the plasma samples from the animals, were diluted 10× in acetone before centrifugation; this has the dual effect of both precipitating proteins and other macromolecules, thus cleaning up the sample, and making sure all cabazitaxel is solu-bilized. Internal standard (hexadeuterated CBZ) dissolved in acetone was added to a final concentration of 10 ng/ml during the acetone di-lution to correct for possible matrix effects.
Three commonly used breast cancer cell lines  were used. The MDA-MB-231, (triple negative; Claudin low), was cultured in RPMI 1640; the MDA-MB-468 (triple negative; basal) and the MCF-7 (luminal
A)  cell lines were cultured in DMEM. All medium was fortified with 10% (w/v) fetal calf serum albumin (Sigma) and 100 units/ml penicillin/streptomycin (PenStrep®, Sigma). All cell lines were obtained from ATCC and were routinely tested for mycoplasma. Cells growing in 24- or 96-well plates were incubated with serial dilutions of PEBCA-CBZ, CBZ (non-encapsulated CBZ) dissolved in Tween-80 (Fluka)), and PEBCA without CBZ for 24, 48 or 72 h at 37 °C in an atmosphere of 5% CO2. The toxicity was assessed either by the commonly used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell via-bility assay, by measuring cell proliferation based on [3H]thymidine incorporation, by measuring protein synthesis by incorporation of [3H] leucine and by measuring ATP levels using CellTiter Glo® .