2.7. Characterization of Materials

¹H NMR spectra of all synthesized samples were collected at a temperature of 298 K on a Varian U400 MHz spectrometer.

Size exclusion chromatography (SEC) was used to determine the molecular weight and polydispersity index (PDI) of MPEG-PHAs. MPEG-PHAs were dissolved in DMF and performed on a system equipped with an isocratic pump, a DAWN HELEOS multi-angle laser light scattering detector (MALLS) detector, and an Optilab Rex refractive index detector. The MWs of polymers were determined based on the dn/dc value of each polymer sample calculated offline by using the internal calibration system processed by the ASTRA V software (Wyatt Technology, Santa Barbara, CA, USA).

The stoichiometry of complex of ss-DOX with β-CD in DI water/DMF (19:1) was determined by the continuous variation method, analyzing the change in fluorescent intensity of ss-DOX, where the total concentration of ss-DOX and β-CD were kept at 50 μM. Stoichiometry of the complex was calculated according to the following formula.

The drug release profiled were performed at 37 °C in both mimetic physiological and lysosome conditions: pH = 7.4 and 5.3 buffer solutions with and without DTT (10 mM). 0.5 mg DOX-loaded MPEG-PHA micelles (P-DOX) or ss-DOX cross-linked MPEG-PHA micelles (PSSD) were dissolved in 1mL different buffer solutions and placed in a dialysis bag with a molecular weight cut-off (MWCO) of 3.5 kDa. Then the dialysis bag was immersed in 30 mL of the different release medium and incubated at 37 °C. Samples (2 mL) were periodically sucked up at different time intervals. Meanwhile, the same volume of fresh medium was filled. The amount of released DOX was then analyzed with UV/Vis spectroscopy (Lambda20, Perkin Elmer, Inc., Waltham, MA, USA) at 500 nm. The drug release studies were performed in triplicate for each of the samples.

The concentration of P-DOX and PSSD in these aqueous solutions are: 0.4 mg/mL. Average hydrodynamic diameters were recorded on Zetasizer Nano S (Malvern Panalytical Ltd., Malvern, UK).

TEM studies were performed with a JEOL 2010 instrument operated at 200 kV. The samples of PCCD and PSSD were treated with DTT (10 mM). DTT (1.5 mg) was dissolved in PCCD and PSSD solution stirred for 2 h. A drop of PCCD/PSSD with or without DTT treated aqueous solution was added onto the surface of grid and stayed for 30 min. The excess water was sucked by filter paper from the edge and negative stained by Uranylacetate (2%, w/w) aqueous solution for 1 min and removed by filter paper.

The cellular uptake of DOX-loaded PD micelles (P-DOX), PCCD and PSSD were analyzed by CLSM. HeLa cells were seeded in six-well plates with a cell density of 2 × 10⁵ cells per well in 6-well plate and cultured in incubator at 37 °C overnight. After that, the culture solutions were replaced by free DOX, DOX-loaded PD micelles (P-DOX), PCCD and PSSD DMEM solutions at a final DOX concentration of 10 μg/mL and incubated for additional 0.5 h and 2 h. Then, the culture medium was removed and cells were washed with PBS and fixed with 4% formaldehyde for 30 min at room temperature. Finally, the cells were rinsed with PBS and stained with 2-(4-amidinophenyl)-6-indole-carbamidine dihydrochloride (DAPI) (2 μg/mL) for 10 min. The slides were mounted and observed by an LSM 700.

HeLa cells were seeded on 12-well plates at 1 × 10⁵ cells/well and cultured for 24 h at 37 °C. Then the culture medium was replaced by DMEM (500 μL) into which DOX loaded micelles (PCCD, PSSD) or free DOX were added (10 μg DOX/mL). After incubation at 37 °C for another 1 h, cells were washed with cold PBS for three times before collected and then subjected to flow cytometry analysis. Cells without DOX treatment served as the negative control.

The in vitro anticancer effect of DOX-loaded PD micelles (P-DOX), PCCD and PSSD were measured by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (MTT assay). HeLa cells with a density of 4.0 × 10³ cells/well were seeded into a 96-well plate and cultured in incubator for 24 h. Then the incubated medium was replaced by treated with GSH (10 mM in DMEM) and incubated for another 12 h using without pretreatment cells as the control. After the GSH was removed, PSSD diluted in DMEM (100 μL) were added to cells and cultured for additional 48 h. The culture medium in control were removed and replaced with 100 μL fresh medium containing serial dilutions of free DOX, PCCD and PSSD respectively. Thereafter, 15 μL of 5 mg/mL MTT assays stock solution in PBS was added to each well and incubated for another 4 h. After discarding the culture medium, the obtained purple formazan crystals were dissolved in 100 μL per well DMSO and the absorbance was measured at a wavelength of 570 nm.