computed and recorded three times (*n* = 3). 4.6.2. Drug Entrapment and Loading in NPs

4.6.2. Drug Entrapment and Loading in NPs Erlo and Dox entrapment in the Dox–Erlo NPs was evaluated by the centrifugation of the formulation at an elevated speed of 15,000× *g* rpm at 4 °C for 30 min (C24, REMI Refrigerated Centrifuge, Mumbai, India). The amount of un-incorporated drug was estimated by reading the absorbance of the supernatant at 342 nm and 480 nm using a UV-Erlo and Dox entrapment in the Dox–Erlo NPs was evaluated by the centrifugation of the formulation at an elevated speed of 15,000× *g* rpm at 4 ◦C for 30 min (C24, REMI Refrigerated Centrifuge, Mumbai, India). The amount of un-incorporated drug was estimated by reading the absorbance of the supernatant at 342 nm and 480 nm using a UVvisible spectrophotomer.

visible spectrophotomer. The % entrapment efficiency and the loading of drug were estimated using following equation:

% *Entrapment e f f iciency* = *Total amount o f drug* − *amount o f drug in the supernatant Total amount o f drug* <sup>×</sup> 100 (1)

#### 4.6.3. High-Resolution Transmission Electron Microscopy (HR-TEM)

The morphological characterization of the nanoparticles was studied using a JEOL, JEM 2100 Plus, (Japan) operated at 80 to 200 kV at an ultra-high resolution (UHR). The re-dispersed nanoparticles (0.5 mg/mL) were sonicated for 1 min by dispersion in water. Further, one drop of nanoparticles was stretched over a permeable film grid and dried for ten minutes. Microscopic images were observed and captured at 80 to 200 kV.

#### 4.6.4. Fourier Transform Infrared Spectroscopy (FT-IR)

The FT-IR spectra of Erlo, biopolymer, PVA, Dox–Erlo NPs, and Dox–Erlo-NP conjugates were characterized by FT-IR (Tensor 37, Bruker, MA, USA). Sample of weights of 5 mg were directly placed into the light-beam path and spectra were recorded in a scanning range of 4000–400 cm−<sup>1</sup> .

#### 4.6.5. Differential Scanning Calorimetry (DSC)

The DSC technique was used to compute the melting point and physical state of the drugs Dox, Erlo, biopolymer, PVA, Dox–Erlo NPs, and Dox–Erlo-NP conjugates by using DSC (Pyris 4 DSC, Perkin Elmer, Waltham, MA, USA). This technique estimated the difference in temperature between a test sample and a reference as a function of the time and temperature when the samples underwent temperature scanning in a range of 50–350 ◦C in a controlled atmosphere.

#### 4.6.6. X-ray Diffraction (XRD)

XRD analyses of Dox, Erlo, biopolymer, PVA, Dox–Erlo NPs, and Dox–Erlo-NP conjugates were characterized by a PAN analytical X'pert PRO, (Netherland) working at 40 kV, 30 mA, and 2-theta angle ranges (0◦ to 80◦ ) using monochromatic CuKa-radiation (k = 1.5406 Å).
