Nano instrument

Electrophoretic measurements were performed
with a Zetasizer Nano Instrument (Malvern), equipped with a 4 mW He–Ne
laser (633 nm wavelength). The obtained electrophoretic mobility (u) values were converted into zeta potentials (ζ)
with the Smoluchowski equation^{40 (link)} where ε is the dielectric
constant of
the medium, η is the dynamic viscosity of water, and ε₀ is the dielectric permittivity of the vacuum. The product
of the inverse Debye length and the radius of the particles was 17
at 1 mM ionic strength (the lowest ionic strength used), which justifies
the use of the Smoluchowski equation. For the determination of electrophoretic
mobility of the particles, appropriate volumes of salt solutions and
water were mixed to obtain the desired ionic strength. Then, the BNNS
particles were added from the stock suspension, leading to a particle
concentration of 5 mg/L and a final volume of 2 mL. The samples were
allowed to rest for 2 h at room temperature before each measurement,
and the equilibration time in the device was 1 min. The experiments
were performed in omega-shaped plastic cuvettes (Malvern). The reported
values were the average of five individual measurements, and the average
error of the determined ζ-potentials is about 2–5% depending
on the magnitude of the particle charge.

The measurement of hydrodynamic diameter values was conducted using a Zetasizer Nano instrument (Malvern, UK) equipped with a 4 mW He-Ne laser operating at 632.8 nm. Correlation data were fitted using the cumulants method to the logarithm of correlation function, yielding the diffusion coefficient. The backscattered light was detected at 173°, and the number-average hydrodynamic diameter was calculated using the Stokes-Einstein equation. For the measurement of zeta-potential, the same instrument with laser Doppler velocimetry and phase analysis light scattering was used. All light scattering measurements were repeated at least five times at 25 ± 0.1 °C, and the data acquired were processed using the Malvern Zetasizer software.

Dynamic light scattering measurements were performed on a Zetasizer Nano instrument (Malvern, Herrenberg, Germany). Polymer solutions (about 2 g/L) were prepared in Na₂HPO₄ 0.1 M and CaCl₂ 20 mM and filtrated through 0.45 μm pore-sized filters. The polymer concentration chosen is between 7 and 35 times smaller than the overlap concentration estimated from the intrinsic viscosity. Moreover, additional DLS measurements were carried out at lower concentrations (about 0.5 g/L) and results are shown in the Supplementary Materials (Figure S3). The samples were stabilized at constant temperature for 1 min prior to measurement. All the measurements were carried out at a temperature of 25 °C. Examples of the correlation functions of PCEs can be found in the Supplementary Materials (Figure S4).
The values of the hydrodynamic radii were calculated from the decay times using Stokes–Einstein equation: R_H = k_BT/6πηD where k_B is the Boltzmann constant, T is absolute temperature, η is the solvent viscosity and D the coefficient diffusion.

The particle size distribution of prepared CS-derivatives/drug encapsulated nanoparticles was determined by dynamic light scattering (DLS) using a Zetasizer Nano Instrument (Malvern Instruments, NanoZS, ZEN3600, UK) operating with a 532 nm laser. A suitable amount of nanoparticles was dispersed in distilled water, creating a total concentration of 1%, and was kept at 37 °C under agitation at 100 rpm.