Nanoparticle Characterization

A volume of 20 µL of nanoparticle suspension was diluted in 2 mL of distilled water, then the particle size and zeta potential were measured before lyophilization by dynamic light scattering (DLS) using Zeta sizer Nano-S and Nano-Z from Malvern Instruments Ltd (Malvern, Worcestershire, UK). The values were expressed as median diameter (D 50%) and millivolt (mV), for particle size and zeta potential, respectively. The polydispersity index (PDI) of the particle size was also reported.

Encapsulation efficiency was measured indirectly by quantifying the amount of total un-encapsulated TQ using high-performance liquid chromatography (HPLC) with a validated analytical method as reported before.³⁵ Briefly, after nanoparticle fabrication, 2 mL of the nanoparticle suspension was collected and the supernatant was obtained by centrifugation. The HPLC analysis was performed using Shimadzu LC-20AT equipment (Shimadzu, Japan). A mixture of acetonitrile and water in the ratio of 60:40 was used as a mobile phase at a flow rate of 1 mL/min using Inspire C18 (4.6 x 250 mm, 5 µm) analytical column. The detection was performed at a UV wavelength of 254 nm using a diode-array detector. The encapsulation efficiency was calculated based on the Equation below (Eq.1):

where is the initial TQ concentration used in the oil phase and is the final TQ concentration in the aqueous phase.

The nanostructure of the particles was observed under a scanning electron microscope (SEM; Zeiss, Evo 50, Germany). The samples were sputter-coated with gold before observation under the SEM. The highest magnification images were obtained to observe the freeze-dried nanoparticle prepared from the optimized formulation.

Five milligrams of the optimized freeze-dried nanoparticles were suspended in 5 mL of phosphate buffer saline (PBS pH 7.4) and incubated at 37°C. At predetermined time points (0 h, 1 h, 3 h, 21 h. 42 h, and 1 week), 1 mL of the release medium was removed, and the NPs were separated by centrifugation. Fresh PBS was added to replace the taken amount. TQ in the release medium was evaluated using HPLC as mentioned before.

DSC analysis of individual components of the nanoparticle formulation (PLGA polymer, TQ, Tween 80, PVA and chitosan) and the physical mixture of polymer + drug (1:1) was performed using PerkinElmer DSC-7^® (PerkinElmer, Inc., MA, USA). Equal weight of about 5 mg of each sample was loaded into 40 µL standard aluminum crucibles then heated under continuous nitrogen purging (20 mL/min) at a heating rate of 10°C/min to 350°C. An empty crucible served as a reference.

TQ, PLGA, PVA, TQ-PLGA NPs were examined for FTIR spectra in the range of 400 to 4000 cm⁻¹ at 4 cm⁻¹ resolution (Frontier Optica, Perkin Elmer, Pittsburgh, Pennsylvania, USA).

Three types of stability assays were performed on TQ nanoparticle formulation and TQ solution. The first assay was based on the observation for changes in the particle size, PDI, and zeta potential using Malvern Zetasizer Nano-S and Nano-Z (Malvern Instruments Ltd, Malvern, Worcestershire, UK). The physical stability study was performed 1 month after preparation of the TQ-loaded PLGA nanoparticle suspensions at four different storage temperatures (−40°C, −20°C, 5°C and 25°C).

The second stability assay involved the assessment of nanoparticles in the cell culture medium. Here, the nanoparticle suspensions were added to complete cell culture medium (DMEM) at different concentrations (0.1 mg/mL to 10 mg/mL). The nanoparticle suspensions were incubated at 37ºC and were tested after 24 h and 48 h, respectively. The parameters used included changes in the particle size, PDI, and zeta potential.

The third stability assay involved the assessment of chemical degradation of TQ in complete cell culture medium (DMEM) using the HPLC.

A375 human melanoma cells were obtained from American Type Cell Culture (ATCC), Manassas, USA. The cells were cultured in T75 and T25 flasks (Eppendorf, San Diego California, USA) at 37°C and 5% CO₂ using High glucose Dulbecco’s modified Eagle medium (DMEM) Eagle’s Minimum Essential Medium (EMEM). Fetal bovine serum, penicillin-streptomycin, and HEPES buffer were added to a final concentration of 10%, 2%, and 1%, respectively, to complete the growth medium.

A375 cells were seeded in 96-well flat plate at a concentration of 1 x 10⁴ cells/100 µL/well. For concentration-dependent studies of the cell uptake, after the cells adhered to plate within 24 h then treatment started with 0.1, 1.0, 2.5, 5, and 10 mg/mL concentrations of coumarin-TQ-loaded PLGA NPs for 24 h. Meanwhile, for time-dependent cell uptake, the cells were assessed after 2 h, 6 h, and 24 h of treatment, respectively, using a fixed concentration of the NPs. On the time of assay, the cells were washed gently with ice-cold PBS three times before being detached by triple E in the incubator for 2 min.³⁶ The cells were centrifuged at 800 g for 4 min and the supernatant was discarded. The cells were then re-suspended in 4% of formaldehyde for cell fixation and incubated in ice for 10 min. The cells were centrifuged and re-suspended in PBS buffer saline before being evaluated with the flow cytometer. The forward light scatters (FSC) were used to count the total number of cells after the deduction of the background caused by some free NPs in the medium. On the other hand, the green fluorescence channel was used to count the positive cells (successful cell uptake if fluorescent NPs).

The efficiency of cell uptake was also qualitatively assessed using a fluorescent microscope (Cytell cell imaging system, GE Healthcare life science, Buckinghamshire, UK). In this assay, the A375 cells were seeded on round-coated coverslip mounted in 24-well flat plate at a concentration of 1 x 10⁴ cells/well. Cells were allowed to attach for 24 h and then the cells were incubated with a suspension of 1.0 mg/mL coumarin-loaded nanoparticles in growth medium for 2 h. The coverslips were then transferred into wells containing ice-cold 4% paraformaldehyde for 10 min fixation. Then the coverslip was lifted and placed on top of the glass slide with cells facing upwards. A 50 µL of mounting medium containing fluorescent dyes of DAPI and phalloidin with the ratio 1:1 was dropped on the cells before being covered by square coverslip cleaned with 70% ethanol. The glass coverslips were sealed to retain the position on the glass slide before being examined under the Cytell fluorescence microscope.