Mastersizer 2000

An aliquot (1 mL) of reconstituted (5% (w/v)) protein extract adjusted at pH 7.0 was pipetted into a Mastersizer 2000 (Malvern Instruments, Malvern, Worcestershire, England) equipped with a Hydro 2000S sample dispersion system (set at a stirring speed of 1000 rpm) interfaced with Mastersizer 2000 software (version 5.61; Malvern Instruments, Malvern, UK). The volume weighted mean (D[4, 3]), the surface weighed mean (D[3, 2]), the absolute deviation from the median (uniformity) as well as the average particle size (d(0.5)), the sizes of particles below which 10% and 90% of the sample lie (d(0.1) and d(0.9), respectively) and the specific surface area (SSA) were recorded. The particle refractive index and the continuous phase refractive index used were 1.52 and 1.33, respectively. The beam length was 2.35 mm and the minimum detection limit was 0.02 µm. Measurement integration time was 12,000 ms. The analysis was performed in triplicate with three measurements during each run.

The zeta potential and chemical state of the GO (1 mg/mL in deionized water, room temperature) and AFGO (1 mg/mL in deionized water, room temperature) were analyzed by a zeta potential analyzer (Mastersizer 2000, Malvern, UK) and fourier transform infrared spectroscopy (FTIR) (Tensor 27). The particle size of the AFGO, GG microsphere and GG/AFGO–Dox microsphere was measured by a laser diffraction particle size analyzer (Coulter LS230 and Mastersizer 2000, Malvern, UK). The morphology of the GG/AFGO microsphere and GG/AFGO–Dox microsphere were characterized by SEM (Jeol JSM-6400, Tokyo, Japan). The elemental composition was performed by energy-dispersive X-ray spectroscopy (EDS).

The particle size of the prepared PLGA microspheres was measured using a Mastersizer 2000 (Malvern Instruments, Malvern, UK), based on a laser diffraction method. The volume mean diameter (VMD, μm) was obtained as the representative value of particle size. Further, the PSD was estimated in terms of the SPAN value. The SPAN value was calculated by dividing D_90% – D_10% by D_50%; generally, a smaller SPAN value means a narrower PSD.

The distribution of particle sizes was determined using laser diffractometry with a Mastersizer 2000 containing a Hydro 2000S module (Malvern Instrument, Malvern, UK) for wet sample dispersion. Particle sizes are expressed as the volume mean diameter (VMD) in micrometers (µm). The size distribution was determined using the apan value, which is calculated as the ratio of D90%–D10% to D50%, where DN% indicates the volume particle diameter at each cumulative volume percentage. Smaller span values represent narrow particle size distributions.

Droplet size distribution of the emulsions was carried out by the laser diffraction method in a Master sizer 2000 (Malvern Instruments, Malvern, Worcestershire, UK). Average Sauter diameter (D_3.2) (Equation (2)) and polydispersity through the dispersion index (Span) measurements have the assessment of the variations in the droplet sizes for a given emulsion (Equation (3)).
$D_{3.2}={\displaystyle \sum n_i{d}_i^3}/{\displaystyle \sum n_i{d}_i^2}$
where n_i is the number of particles with diameter i (d_i).
$Span=(d(90)-d(10))/d(50)$
where d(90), d(50), and d(10) represent the diameters to the cumulative distribution at 90%, 50%, and 10%, respectively.
Zeta potentials (ζ) of the CH, hydroxypropylmethylcellulose, MG, and emulsions were determined in a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK). pH was adjusted in a pHmeter (pH210 Hanna Instruments, Woonsocket, RI, USA) equipped with an electrode (HI 1332 Hanna Instruments, Woonsocket, RI, USA). Emulsion stability was determined by measuring the upper oil-rich layer formation after leaving it to stand in 30 mL of each emulsion in conical graduated screw cap tubes and stored upright at 25 ± 1 °C. Formation of upper cream-like layers was observed every 15 min for the first 9 h, and then every 24 h for two weeks and then once per week.

The Mastersizer 2000 (Malvern Instruments Ltd., Worcestershire, UK) was used to monitor particle size and particle size distribution by preparing MP solution. The MP dispersions were diluted to 1 mg/mL with deionized water to avoid multiple scattering. One milliliter of MP solution was added into a clear zeta cell. Size measurements were reported as the volume-weighted mean diameter (d_4,3) (Equation (1)) and surface-weighted mean diameter (d_3,2) (Equation (2)). The indexes of protein were analyzed using the Malvern Mastersizer software (version 5.12c, Malvern Instruments Co. Ltd., Worcestershire, UK) [15 (link)]
$$d_{4,3}=\frac{\sum n_i{d}_i^4}{\sum n_i{d}_i^3}(\mu m)$$
$$d_{3,2}=\frac{\sum n_i{d}_i^3}{\sum n_i{d}_i^2}(\mu m)$$
where n_i is the number of droplets of diameter d_i.