3.4.1. Analysis of Zeta Potential, Particle Size, and Transmission Electron Microscopy (TEM)

The developed nanoparticles, namely DPLGA and ADN-PLGA, were characterized for particle size using photon cross-correlation spectroscopy. The formulation sample was put in a clear polystyrene cuvette (path length = 1 cm) after being diluted with double distilled water to ensure that the light scattering intensity remained within the instrument's sensitivity range. Size measurements were performed using a nano-size analyzer (Nanophox, Sympatec India Pvt. Ltd., Mumbai, Maharashtra, India) at ambient temperature [33]. Zeta potential was measured on a zeta meter (Delsa Nano C, Beckman Coulter, Tokyo, Japan) [34]. For surface topography, images of nanoparticles were captured using high-resolution TEM (JEM 200, JEOL, Tokyo, Japan). The nanoparticle dispersion was put on a carbon-coated formvar grid and stained with neutralized phosphotungstic acid (1%) before being imaged under a microscope [35].

#### 3.4.2. Entrapment Efficiency (EE, %)

EE corresponds to the percentage of DTX encapsulated within or/and adsorbed onto the DPLGA and DTX-DPLGA nanoparticles. The nanoparticles suspension was centrifuged at 5000 rpm for 5 min to settle down the precipitated drug [36]. The supernatant was collected and centrifuged (Sorvall benchtop centrifuge, ThermoScientific, Mumbai, Maharashtra, India) further at 21,000 rpm for 30 min at 4 ◦C to settle down the nanoparticles. The concentration of DTX in the supernatant and precipitate was calculated using the previously reported and validated RP-HPLC method [7].

#### 3.4.3. In Vitro Release of DTX in Buffers

The release of DTX was studied from pristine DTX, DPLGA nanoparticles, and ADN-DPLGA nanoparticles by suspending in a Float-A-Lyzer (G2, Spectrum, Repligen, MA, USA) in two different release media, namely phosphate buffer saline pH 7.4 and sodium acetate buffer pH 5.0 containing Tween 80 to maintain sink condition and facilitate release [37]. The tests were performed at 37 ◦C (n = 6). The dialyzers were placed in sealed beakers with 100 mL release media and stirred on a magnetic stirrer at 100 rpm. DTX released at different time intervals was analyzed using the validated RP-HPLC method at pre-determined time intervals by withdrawing 0.5 mL of release media over 5 days. Immediately after sampling, the volume of release media was maintained at 100 mL by replacing equal amounts of release media. Release media samples were filtered through 0.22 µm

PVDF filters (Millex-VV, 13 mm, Merck, Mumbai, Maharashtra, India) and analyzed after appropriate dilution with a mobile phase of the RP-HPLC method. The dissolution profiles were compared using the *f* <sup>2</sup> similarity factor.
