Characterization of Drug-Loaded Nanoparticles

Drug content of the nanoparticles was determined by HPLC analysis using a Shimadzu HPLC (model LC-20AD). Supplementary Figure 2 displays the HPLC profiles of four standard Paclitaxel solutions.

Nanoparticles (3 mg) were added to 50mL of water/acrylonitrile (ACN) (50/50 v/v) and stirred with a magnetic stirrer until complete dissolution. A clear solution was obtained, which was filtered through a hydrophilic 0.45μm PVDF filter (since Paclitaxel is hydrophobic) and assayed for drug content by HPLC. The column used was a Eclipse XDB-C18, 5μm, 250 x 4.6 mm. The flow rate was 1 mL/min and the column temperature was 25°C. A diode array detector was used at 227 nm, and quantification of the API (Active Pharmaceutical Ingredient) was based on a calibration curve created by diluting with mobile phase a stock solution of 20 μg/mL Paclitaxel in water/ACN (50/50 v/v) to concentrations 20, 10, 5, 2.5, 1 and 0.5 μg/mL (Supplementary Figure 3). Nanoparticle yield, drug loading and drug entrapment efficiency were calculated from Equations (1)–(3), respectively:

Morphology of the prepared nanoparticles was examined with a Scanning Electron Microscope (JEOL, JMS–840). Samples were coated with carbon black to avoid charging under the electron beam. Operating conditions were: accelerating voltage 20 kV, probe current 45 nA, and counting time 60s.

Particle size distribution of the Paclitaxel/polyester nanoparticles was determined by Dynamic Light Scattering (DLS) using a Zetasizer Nano instrument (Malvern Instruments, Nano ZS, ZEN3600, UK) operating with a 532 nm laser. Nanoparticles were dispersed in distilled water at 1%w/v and kept under agitation at 37°C in a water bath. Particle size was measured at different time intervals after sample introduction into the dispersal medium. All measurements were performed in triplicate and the results were reported in terms of mean diameter ±SD.

Lyophilized PPSu-PEG and PPSu-PEG-Paclitaxel NPs were suspended in PBS buffer (NaCl 137mM, KCl 2.7mM, Na₂HPO₄ 10mM, K₂HPO₄ 2mM) with pH 7.4 and handled as described above to ensure even distribution. Two rapid washes with PBS followed in order to remove any weakly bound drug remaining on the surface from the encapsulation reaction that could result in a burst effect.

The concentration of both nanoparticle types was calculated as polymer mass per volume (1.683 mg/mL) and the drug concentration was determined by the quantity of drug encapsulated in the polymer. Suspended NPs were incubated at 37ºC, with stirring at 100 rpm. At specific time intervals, the suspension was centrifuged (6000g for 10min) in order to separate the NPs (pellet) from the solution containing the released drug. A small sample volume (20μL) was removed from the supernatant of both PPSu-PEG-Paclitaxel and PPSu-PEG (used as control) at each time point and assayed in order to determine the amount of the released drug. The detection and quantification of the released drug was carried out using fluorescence spectroscopy (Infinite M1000, Tecan), with a suitable quartz cuvette, with the following parameters: excitation from 240nm, emissions scan from 280nm to 450nm, 150 flashes (400Hz) and 17nm bandwidth. The experiment was performed in triplicate.